addendum - shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/951/18/18... · 2012. 7. 11. ·...

Addendum

Based on this thesis work four papers were published in peer-reviewed international journals, one paper

war presented in national conference. and one national newspaper has cited one of the published works.

Parija SC, Khairarr K. Entamocba moshkovskii and Enfamoeba dispar-associated infections

in pondichcrry. India. J Health Popul Nua. 2005 Sep;23(3):292-5.

Parija SC. Khairnnr K. Detection of excretory Enfameha hisrolyica DNA in the urine, and

detecticm of E. hisrolyfica DNA and lectin antigen in the liver abscess pus for the diagnosis of

amoebic liver abscess. BMC Microbial. 2007 May 18:7:41.

Kbairnar K. Piarija SC. A novel nested multiplex polymerase chain reaction (PCR) assay for

differential detection of Enramorba hisrolyrica. E. moshkovrkii and E. dispar DNA in stool

sample,. BMC Micmbiol. 2007 May 21;7:17.

Kbairnar K. Pyija SC. Palaniappan R. Diagnosis of intestinal anmebiasis hy using nested

polymcrar chain rcact~on-restriction fragment length polymorphism assay. 1 Gastrwnteml.

2007 Aug:42(8):63I 40. Epuh ZOO7 Aug 24.

Kbairnnr. K. Parija. SC. A novel nested muldplex PCR assay for differential detection of

Enrmoeho hisrolvricu. E. moshko~~rkii and E. dispar in stool samples. In: Ahsfracts of the I V

Winter Symposia on Molecular lnsighrs into Digestive Disorder. Christian Medical College.

h e m h e r 15'- 16". ZOOS, Vcllorc. India.

Common disea\e, sirnple test. In: Know how. The Telegraph Calcutta. August 6". 2007.

Kolkau. India.

N.B: Reprints of the publicorians enclosed

J HEALTH POPUL NUTR 2W Scp;23(3):292-295 0 2905 ICDDLB: Cmtre for Health ud Population Rncnrch ISSJ'iJ-7 S 5 . W . 2 0

SHORT REPORT

Entamoeba moshkovskii and Entamoeba dispar- associated Infections in Pondicherry, India

Subhash Chandra Parija and Krishna Khairnar

Departmen! of Microbiology, Jawaharlal Institute of Postgroduafe Medical Education and Research, Pondicheq 605 006, India

ABSTRACT

The prevalence of Larcdo stra~rcEnfamoeba moshkovskii-and non-pathogenic E dispar in patients attending the Jawaharlal Institute of Postgraduate Medical Education and Research hospital, Pondicheny, India. is-reported k c . E, mo.shkov.rkii is reported for the first time in India. The species an morphologically indistinguishable from pathogenic E hisaivfr~~. Of 746 stool samples screened, 68 showing cyst or lrophomi~e stage of E. hirlolyficu, E. dkpur, or E, moshkovskii were subjected to small sub unit (SSU) rRNA gene-based polymerase chain reaction, which revealed a higher prevalence of E. dirpar (8.8%) and E, moshkov.sklr (2.2%) compared to E hisfolyfica (1.7%) in patients. Only 1Yh of the 68 slwl samples, resembling E. his!olyricu by microscopy, were actually E. hirtolytica, implying that HI% of suspected infcctions were misdiagnosed and would have been treated unnecessarily with anti-amwhic drugs.

Key MI& Enramorha hislo!~r~ca: Enramwho moshkovskii; Entawwba dispur; Amwbiasis; Diagnosis, Laboratory; India

INTRODUCTION polymcraw cham &on (PCR). In the clinical setting, this may lead to a misdiagnosis and unnecawy treat-

E ~ b u ~ i b ~ p i m a n l ~ a bee-livingImcrba ment chemotherapy (3). I t is indistinguishable in its cyst and ~ m i t c forms from E. hirrolytica, the causative agent of mabiasis. E. mathkovskii has so far rarely bnn shown to infect MATERIALS AND METHODS

h m m (I). E. marhkowkii in humans has boen repolred hun No& America, Italy. South Afhca, and Banghksh, but it bas never b a n associated with disease (2). The prevalcncc of E machsk i i in India has not brm rc-

arlier. The morphological similarity of E. m h - kowW and E. dirpm to d isu~sausing E. histolyfica nuka it important to differentiale the h c c specin by

('ampadmu and rcpint nqurm should be dmcucd lo: u subbuh Chuldm Rnja DirtxtOf-RoTcrra d Hed Uepmnan of Microbiology J a u r W l l lMim of Poltpndtu(e MWEhrvianndueaalch Pondihmy M5 006 lndi hail: [email protected] Fu: 91413-2272067

Stool spahncnr, for this study were obtaioed k m patients attending the Jawaharlal Institute of Postgraduate Me- dical Education and Research (JIPMER) hospital, Pon- dicheny, India lo total. 746 stool samples from patients clinically suspected la have gasmintathral infections were collected during July-December 2004 and w m screened by minoseopy. of which 68 showing popb zoitdcyst were subjected to E. hisfobtica, E. dirpor. and E mnrNovskii-specific mted PCR.

The DNA was isolated using the rrtyltrimethylam- monium bmmidc (CTAB) utmctiion mahod (4). The extracted DNA was psssed through DNA clean-up spin columns (Ehgdm Gmei. Bangalm) to remove PCR inhibitors. Bwd on the sequences of h e small subunit

(SSUtrDNA of E. hwtolylca and E, dispar, nested sets of primers (designated E-liE-2, Eh-l/Eh-2, and Ed-IIEd-2) were used for dcieaing E. hictolyrca and E, dispar in s ~ l specimeos. The PCR was given a hot stBlt by pre-incubating the PCR mix at % "C for two mlnutes, followed by 30 cycles--each consisting of 92 T f 0 r ~ , 4 3 " C f o r 6 0 s , a n d 7 2 ~ C f o r 9 O s a n d 7 2 ~ C for five minutcbonc cycle for the final extension (5). In the ncitcd PCR, d i n g temperature was Riscd to 62 OC, leaving thc ~ ~ I C I pMKtcfl of the ~~plif%atim cycles unchanged. E. hutolytica and E. di~pcu-specific nested SSU-rDNA gene amplification products w m double-ctiptcd with mbictim m d o n u c k &el and Sou96-1 for two hours a! 37 "C according to the insuuc- ~ l m c of Ihe manufacturer ( B a n g a h Genei) to verify ~hc identity of species. W u c t s wcre visualized on a 1.3% agarosc gel conraining ethidium bromide (0.2 mg/mL).

The product of ncsted PCR from both E histo1ytic.a and E drrpor showed 900-bp fmgments which were further confirmed by restriction fragment length polymorphism (RFLP) The RFLP pattern for E hirrolvricu 5howed 550-bp and 350bp fragments and undigested 900 bp. whercas for E. &par it showed 700 bp. 550 bp. and conflwnt bands of 200 bp and 150 bp (Fig. I).

. . .-.- - .--.. ~. . .- . "- ~i i . I . Enrumcrho huo!aicu and E ~ i r p r - s ~ c c i f i ?

nc~ted SSU-rDNA PCR products. Odd- and/ cven-nwnbd Ipm n p m t undigested and; Uru-land SuuPb-I-digalal PCR pdwa re*, DCC~IVCIV

Based on the sequence of the SSU-rDNA gene of E. m h s k i i Lado (GcnLkk sccesgioa m. AF 149906). a nested x t of primers (designated Em-1Em-2 d nEm- ImEm-2) was uscd (6) f a detecting E. moshkovskii in stool DNA.

The PCR conditions were ssmc m described above, except that the annealing bmpemtw was 55 O C for the f i r s t P C R u d 6 2 ° C f o r t h e d P C R E . ~ M o v s k i i - specific lYStcd SSU-rDNA gene amplilidoo pmducts w m digested with resbiction endonuclaw XhoI for om hour at 37 'C according to rhc instructions of the manufacturer (Promega) to verify the idmtity of species. Products were visualized m a 1 .go/. agarose gel wntain- ing ethidium bromide (0.2 mg/mL).

The product of nested PCR hwn E. moshkovskii showed a 258-bp fragment whlch was furher confirmed by RFLP. Xhol exclusivcly cuts the 258-bp product to produce 23bbp and 22-bp fragments (The 22-bp product is not visible in p l because it was too s d l to be resolved in I 3% agarosc gcl). Thc standard swin of E. moshkowkii Laredo showed a higher molecular size (300 bp approximately) product compared to clinical isolates (Fig. 2). . . ...

rDNA PCR paluas. Odd- ad even-numbed lanes represent undigested and Xhol-digested PCR d u c t s resoectivelv

mnrbkavsh,, lsaes 718. E marhbvsb, Lucdo. &. A IWbp DNA ladder

DNA from standard cultwrs of E. histo/ytica HM- I : IMSS, E. dirpar SAW760, and E. moshkovskii Laredo was used rr W t i w canml, and stool spmpks showing no hwphozoite/cyst were used as negative control.

RESULTS

The primer sequence for E. moshkovskii. E. histolyfica, d EE, dirpa, blasted m the genome database of all orga- nimv, in the website @apJ//lwww.nebi.nh.nih.govb~, was found to be specific for the study. Moreover, the amplified producu of nested PCR were restriction-digested to rule out my non-specific amplification.

Tk rcfercnce swim-E. moshkowkii Laredc-gave a band al approximately 300 bp with the &. moshkov.rkii. \pccific SSU-rDNA-nested primm, whereas the con- o u L E hirtolvriro HM- I : N S S and E. &par SAW760 DNAs-was negative.

The results of ncstcd PCR-RFLP on the 68 stool DNA samples showng aqhmi tdcys t mcmbling E, hirtoh- 11to are shown in Table I.

One sample, negative by stool PCR for both E. hisrolyfica and E. dirpm, wwas eventually positive for E. mosh- bvrkii. Sixteen of 17 E. mo.rhysk'i-positive stool samples were also positive for E. histolylca, E. &parr or both by SSU-[DNA PCR.

Comparison of SSU-rDNA sequences from E. mosh- kovskiiCii. E. histolytica, and E. dirpor showed that the restriction endonuclease Xhol cut exclusively in the E. mmhkovskii-spaific, 258-bp-ocstcd PCR pmduct to produce 236-bp and 22-bp fragments. Products from all the 17 positive stool samples aad the Laredo sbain showed the preznce of this site (Fig. 2).

The prevalence of E. moshkovskii, E. dispr, and E. hisfolytiro among patienb clinically suspected to have gastrointestinal infections, attending the JlPMER hos- p~tal, is shown in Table 2.

DISCUSSION

Thc study was conducted to identify the prevalence of E. rno.rhkovskii, E. dispar, and E. histolytica in stool

TaMc 1. Summary of Entantoeha dispar. E. hutolytica, and E morhkovskii-specific nested PCR-RFLP results/ on \tool ipecimen\ found m~croscopically posxtl\e for amoeba resembl~ng & huto!b*nco - - . . . - - - -- -- -. . -

lvne of ~nfecl~on No of samplc5 p o ~ t t ~ t c b) nested PCR-RFLP (n=68) 70 of stools pos~ti\e

F d~rpar (mom-mfcct~on) F hrtrolvr~ca (monc~mfcct~on) f mo\hkowkir (mono-mfccrton) t d u p r + E m h h u ~ ~ s k i i (mud) E dupur + E hutolytica (m~xed) t d u p r E hrrrol~tru * E muihkobskii Im~rcd) - -- - - - - - 05 7 3 - PC'R ~oGm-&c c h n -lion. RFLP- Rmnctlon fragment length polymorphism

tic u, and E rnurhkombl among p t~enls at-/ tcndm~ h e JIPMER how~tal c l~n~ul lb ,us- pcctedrto have gaslro~niest~nal lnfeci~ons

/ . _ _-- - - ' Typc of No of .mnples Prevalence i Lnrayehu positive by nested (96) 1 s . ICS PCR-RFLP p.. .. - _ . -- .. -. - - - .. .. ' E. mcrrhkoi,.vkii 17 2.2 E dupar 66 8 8 E hr.ito~t~ca -- 13 1 7 --- -- Toel no of stool samples ~ncludcd m thc study-746

JIPMER-Jawahulal l ~ l ~ t u t e of Poslgraduste Medical Educrtlm and Research

PUR-Polymerase cham maceon RFLP-Rcsmctm fingment length polymorphism

. - _ .. .- _ _

sampln of patients anending the JIPMER hospital. The study. for the first time. reporb the prevalence of E, moshkov.rkii in India.

The morphologtcal simtlarity lea& to confusion in d iaps i s of amoebiasii in clinical &p. We have uxd nested PCR to detect infections due to E. hkrol,.tica. E. hpar. and E nashkorskii ii bsausc PCR i i n c ~ a r s the specificity and is mom efficient in amplifying stool DNA.

Our study included pahenls from varied age-groups and fmm different geographical localities. which shows the wide dismb~tion of E. moshhvskii and E. dirpar in Pondtcherry and its ncighbouring areas.

The shldy has several inlcmsing fd ings . Only one ptimt with dysentery h e d E. marhlovuCii-associated

E nwslkom&ii in Pondkkrry, India

mono-infection. The cause remained undetermined, bacterial actiology by routine s lwl culture was negative. Other investigations, such as viral study, could not be rhmc in the Isbontay. The high prevalence of E, moshkovskii among thc study population supports the view that humans are a hue host for this frcc-living amueba and arc not just transiently infcctcd (6). The study also answers the myslcry of somc microxop~cally-positive but antigenically- and PCR-negative rcsults for E. hlsra- htiru and E. dispar (7). which, in our study, was due to L marbvsfir . Eleven ptients with mued infections duc to E di.rpr and E. moshkor:skii and five patients due to E di.rpor. E hi.rto!vrira. and E. morhkovskii had no diarrhoea or dysentery. bul had complaints of mild gistro~ntestinal discomfofl.

The study has shorn apprec~ably a high prcvnlencc oCE. disprrr and E marhkowkrr in the pntienb ccmpgml lo F: hrrmnl~~tico which reveals thai only 19% of the 68 sttrd samplcs, resembling E hi.vt~~/~'lica by microscopy. ~ c n : wtually E hutol~trcu. implylng that 81% of sus- ~ c l c d infections were mixliagnosd and would have tuxn ucatcd t t n w x s d y w~th MII-omocbic drugs when d l r p ~ s e d bascd on microscopic findrnp alone.

Thus. cpidcmiologic studlrs md clinical diagnosis of E hr.~tol~~rrc~au*;ia~ed infection, which are b a d on n~ t~rph~logra l cumlnatiun alonc, arc prmc to cmr. In ic r~~a~r r~ due to both E. J ~ ~ p a r and E ma.vhkovskir are a\uryia1cd with PIynquOmslK UVDcr stage. Thc lropho- ~uiicr c~fh,th E, dispur and E, mr~shkov.skrr lack the ca- p h ~ l ~ t ) to invak !he inteulnal m u m s mi do n a have :in) ingcstcd erythrocflcs unlike that of E hrsml~trca. P('K I,. thcnfwc. csscntlal to datcngulsh E h ~ ~ m o l ~ ~ ~ ~ c u fic~m E J u p r nnd E w.vhko,:~br

ACKNOWLEDGEMENTS

We s i m c l y thank Dr. C. Graham Clark from London School of Hygiene & Tropical Medicine for providing us with lyophilized DNA of standard cultures of E. histolyfica HM-I: IMSS, E, dispar SAW760. and E. moshkovskii i i d o .

REFERENCES

I. C W C G Diamond LS. lntraspecific variation and phylogmetic relationships in the genus Entumoehu as rcvealed by riboprinting. J Euk Microbial 1997;44: 142-54.

2. Clark C 4 Diamond LS. The Larcdo strain and other Entamwho hirtolyrica-like amoebae arc Enromoeha moshkovskii. Mol Biochem Pararitol 1991:46:11-8.

3 Parija SC. Text hook of medical parasitology (proto zoology and helminthology). 2d ed. Chennai: All India Publishers & Distributors. 2004:Zb-61.

4. Clark C G Diamond LS. Enramoeba hktolylicu: a method for isolate identification. Erp Pura~ilol 1993;7:450-5.

5. Haquc R. Ali IKM, Akther S, Pem WA. lr. Compa- nsun of PCR, isoenzyme analysis. and antigen detection for diagnosis of Entarnwba h~srol,.fica ~nfcction. J Clin Micmhiol 1998;36:449-52.

6. All IK. Hossain MB. Roy S. Ayeh-Kumi PF. Petri WA. Jr.. Haque R. Clark CG Enrumofha mush- kovskii infections in children. Bangladesh. E m q In/ecr Dis 2003;9:580-1.

7 Haque R, Neville LM. Hahn P, Petri WA. Jr Rspld diagnosis of Enmumwho infection by using Entu- mwba and Entamwba hirtolytica stool antlgen detect~on kits. J a n Micmhiol 1995:33: 2558-61

Research article

Detection of excretory Entamoeba histolytica DNA in the urine, and detection of E. histolytica DNA and lectin antigen in the liver abscess pus for the diagnosis of amoebic liver abscess Subhash C Pariia*' and Krishna Khairnart

\Jdrcu Imanmrnt niMirrobioM, lawaharlal lnailulr ol I'ougradualr Mcdlral Wucauon and Rrwarth (III'MER). h d u c h q . India

imn8l: SuDhlrh (: Panla' . pri i lu@nnl urn: Knshna Khairnar . hkha~rnarOyahm cn.in * lilrrrrpondin~ aulhor tl'qual ronlnbutc~n

B.Jct"und: Amotbic Inn abscess (AM) and pyogenK ln&abuesru (~LA)appeii ~defmcal by u k r w w d and other nnagmg tcchn~ques Co4tcclon d Mood or Imr abscess pus for &agmnis d Hver abscesses a an lnmne procedure and the procedure rrqum technlul aperuse and dbpnabk v w Cdkcroon d urlm IS a nmtnwm procedure Therdore, &re has been much ~nfernt s h towards the use of unm a an a h e m m cluval speclmen for the ckapor~s d s a purrmc mfecuons Here we repwr for the fin1 time rhe detecuon oi E haw DNA excreted m the unm (or d l r g a n of dw urn of A!A

Result.: L h6lcfpca DNA was detected m lner abscess pus specimen d 80.4% of ALA patients bv a nested muhi~icx cabmerue c h n ructlon IPCR) mnefnc 1651ike r RNA e m . The nested P(IR & t e n d L'hmoir$o DNA m aW 37 (100$) live; abrcessVwr specimens c ~ l l d pnor to metrondudc treaunenr but *re detected m only 53 of 75 (70 6%) pus rpcornens cdkcred aker thervr mth metromoude Lmlhrh. the PCR detected E hu&nca DNA In 21 d 5 1 0 9 6%) brlne wui;r;ns d ALA mucnts. The teit detected E hmotfl~a DNA in only 4 of 23 ( j 7.4%) urine specunern cdlcnedpnor to r r von ldude treatment. bm were detected in 17 of 30(~6.7%) urme s c e h m cdkcted aker t r uumm rrhh rnetronidude. Thc mnme-imked immunororknt asnu (ELM) lor the detecuon d kctin L h-D antigen In the live;abscas pus showed a smritinr/ d 50% and rhe d i r ec t haemylluonauon (IHA) an fw detection of amoebic antibodwr in the m u m showed a smut iv i i d 76.8% for the d i i i s d the AM.

Cmc*don: The presmt study for the k t time shorn h t rhe kidmy turner ~n ALA patienrs a pemdk to L ~ ~ D N A mdecvle resulting in excretion d f hkW@ca DNA in u r n which c m b e d n m c d b y K R T h c a ~ J l o ~ h t t h e K R l o r ~ o f E h ~ a D N A i n w i n d p l i a n r r ~ A L A c m ~ h o k ~ d a ~ ~ t u ~ r l o ~ e r r t h e c ~ n e o f t h e dbcacr bUomq t h e w by Wonidude. The deteniar d E hiuc+a DNA in urim specimen d A U p c i m a p r o v i d n a m ~ l o r t h e d ~ d A l A

hckgmund to 50 mil l ion symptomatic cases damocbiasis worldwide 'nleclion with Enrn~nabn hamlynra, r-ulu in 34 million each year, causing 40 to 100 thousand deaths annually

Page 1 of 10 f p * * ~ " m ~ c * m p . D u J

111 Mortality from amoebiasis is mainly due t o extra- lorestinal pathology, o f which amoebic liver abfcess (ALA) is the most common. I f leh untreated, A l A can wp - ture into neighboring t i w e and spread t o the brain and orher organs via hematological route producing serious morbidity and mortality. I t is difficult t o differentiate clin- ~cally the AIA from pyogcnic liver a b w m ( P U ) as well as lmm other space occupying lesions of liver such as hvdalid cyst and liver hepatoma 12.31.

lrnaglng techniques like ultrasound. computed tomogra. phy, and magnetic rmnance although are highly sensi- rlvr l o detect absccsm i n the liver o f varied aetiology, however fail to distinguish specifically AIA h o m that o f I'I.4. IPS than one third o f patients wi th AIA have active dtanhea 131. Hence, s t m l microscopy and srool antigen Jctcclion is not very helpful for diagnosis o f ALA. I n fact I w than only 10% of A IA patienw have identifiable E. ~ISICIJ~IKII i n s tm l specimens 141.

laboratoty diagnosis o f A l A is usually established by con. ccnt~onal antibody-based serolog~cal tests. Nevertheless. Ihr matn d~sadvantage with anubody detection is that i m m ant~body levels In individuals living i n endemic ircds. continues to remain por twe w e n for years after ~nirclton w t h E huroiyrua 15-71. l h e demonstration o f ~rnrrbtc antibaltes I n the serum, therefore, fails to

Enumoeba DNA i n liver abscess pw for the diagnosis o f t h e m ~20.21.9.10~.

Collection o f b lood o r liver a b w u pus is an invasive procedure, and the procedures require technical apertiw and disposable *nges 122). The method i f not carried out under stringent conditions is w m d a d with the risk o f acquiring needle-borne infectionssuch as hepatitis Bvirus and human immunodeficiency vinu (HIV). Therefore, o f late much interest has been shown towards the use o f urine as a specimen alternate to the blood for the diagnosis o f some parasitic infections induding malaria, xhist i- somiasis, kala-azar, rystic echinococcosis and neu ro~ i ce rcos i s 1221. Urinary antigen for cystic echinococcosis (CE) and neurqt icercos is has been reported for the f i n t time from our laboratory at Jawaharlal Insti- tuteof Postgraduate Medical fducation and Rerearch (JIP- MER). Puducherry. India 123.241. Our laboratory has developed for the t i n t time a counter-current immunoe- lectrophoresis (CIEP) and co-agglutination (Co-A) t o d a m the hydatid antigen exueted in the urine for the diagnosis o f CE [23,251, and CQ-A IO h e n cysticercus antigen i n the urine for the diagnosis o f neurocysticercosis 1241.

Detection o f DNA i n urine by PCR has been employed for h e diagnosis o f Toxoplasma xondi~. Nesmu xonmhoae.

ilrnote the amoebic ~nfer t ion whether i t is recent o r o ld ~or re l inbu~dm' jn i , ~yrnhorvr ium tubcrculosts; Mytobarre- lunhcrn~orr. wrum amoeb~c antibodies are not demon- num h a e and Chlantvdw rrorhmris infections 126-301. rltrtrd un up to l(Wo of the pattents w ~ t h acute AIA 131

lllc dcrnonstrrtion o f E hu t i t l pa t r ophow iv i n liver ahwtu pus aspirates by microscopy confirms the diagno- r ~ r < t I AIA, but i n k t o f the Iahontories, the amoeb~c Ire phoio~trs can be demonstrated 111 only 15% o f the liver IPU\ (81 Since the t rophozoi~t l o f E, huwlytica are found n i . ~ ~ ~ ~ l y i n the periphery o f the a b w w diagnosis o f A l A by ulturr o f l ~ve r pus for E. hrrtolyr~trr is also unsatisfactory

I J ~ ~ ~ ~ ~ r a s t r a t i o n o f amoehtc ant tgn i n the liver pus IS a ri,trnt dpproach for specific diagnosis o f the ALA. A mon. 'ilr~tinl antibodybased second genenlion I'echLah rnwti~e.l~nkcd inimunonrbent kvuy (EIISA) kit (Blacb- hulk Va ) has been reported to be 78% lcnsitive for the d r t r~~ ion o f E. hululylico l m ~ n antigen i n the liver pus for 1 1 1 ~ dtagnosis o f ALA i n Dhaka. Rangladnh 1101. Studies :glnducted i n various labontor in worldwide including (In have shown that polymerase chain reaction (PCR) is W n s ~ t ~ v c and lpcrific method for detecting Enamoeba "v.4 111 s t w l samples and for diFlcrentrating the morpho- I1arally similar F. h~swlynca From Enumorbd dirplr and '~~urnorbu n r o h k i : 11 1-191. Iiowever, only few studies U"W the PCR have brcn r e p o n d for the dnen ion o f

. . Some studies have also shown that the kidney barrier in rodenu and humans is permeable to DNA molecules large enough t o be anal@ by standard genetic methodologies 131.32). To the best o f w r knowledge 1111 now there is no repon available o n detertion o f Enumoeba DNA in the unne for he diagnosis o f AIA I n the present study. therefore for the first time. we have evaluated a nested multiplex PCR for d n e d o n o f E n u m b a DNA excreted in the urine for the diagnsosis o f U.

Results 'The quantification o f DNA i n the liver abscess pus and urir~e specimen hy Speclrophotomnric analysis showed he DNA yield to be approximately85 and 3 ps jml respectively. The purity o f DNA extract from liver abscess pus and urine specimens was found t o be satisfactory as the value o f ratlo o f readings at 260 n m and 280 n m (OD2J O D ,,,,) was approximately 1.8-1.85.

The squencing result o f PCR product o f E. kisrdyncn horn liver absccss pus and urine specimen showed 99% identi- ties to the sequence deposited in GcnBank, l a c d o n number: =I. The result o f assessment o f competi- t ion o f o h e r non targpt DNA present in liver abscess pus (PLA pus negative for E. hurdynca) and urine (negative control group) specimen with larger D N A showed

expected amplification and no nonspecific amplification nestcd multiplex PCR.

taimation of minimum number o f Entamoebo cells detectable by nested multiplex PCR showed that the detection limit of PCR was found to be appmximately 15 t h~rrolyltw cells as m n 1.5 p1 of template DNA from IDOO E. hislolyficn cells/lW &I o f Tris- ethylenediamine ~etraacetir acid (EDTA) (I€.) buffer produced a positive i~gnal.

Ihe IHA test was positive for serum antibodies in the wmm of 86 (61.9%) o f 139 patients provisionally dial-

bacterial culture. These included Kkbsielh pncumioc (n - 9). Roms species (n = 5). Envrobocm (n - 5). Escherichiu wl i (n = 3) and P- (n - 5) . Ten liver abscess pus specimens showed secondafy infection of ALA with aembic bacteria by Cram's staining and bacterial cul. lure. These included K. pneummiae (n - 3), Enuroboctn (n = 2). E. coli (n - I ) , group B S~~lmmelh species (n - 1). Enm- (n - 1) and Coagulase negative S t a p h y l m ' (n - 2). Such secondany infection o f MA with bacteria has been reported previously in the literature 133,341.

In the present study, a total o f 112 out of 139 (80.6%) provisionally dia-d AU patients were diagnosed as

nost-d as MA. The t a t was p i i r i ve i n a higher number o f h a n d remaining 27 patien& were diagnosed 2 P L 4 by scrurn (71 of 102 [69.6%1) u m p l n of paticnts who had following the aiteria mentioned i n this study elsewhere. rw~~vcd prior treatment wilh metronidaznle than those who had not received any prior treatment with metroni- dml r ( I 5 of 37 140.5%l) and this difference was natisu- tally s~gnificant (XI - 8.53. P - 0.003). Mmonidaznle trralmenl was initiated from a few days to w e n 1 we& bclorr collection of the blood samples in these patients. Iwo (4.6%) out of 43 sen from control cues were posi- tnve for antiamcebic antibody by IHA.

Ihc'SechLb E, hawlyrm II ten was positive for E. hisrolyr- I,,I LaI/CalNAc l d n antigen i n the liver abscess pus o f >I, (40 3%) of 139 provisionally diagnosed ALA patiena lhr l t rh lab F h l rw ly~~a II test detected lectin antigen in

The result o f nested multiplex PCR performed on the liver abscess pus is depicted in Figure I. The nested multiplex PCR test was positive for E. hiuolytiw DNA in 90 (80.4%) of 1 12 liver abxess pus specimens (Table I ) . The nested multiplex PCR could detect E. hawlyncn DNA in the liver abscess pus of all 37 AU patienta (loo%), who were tested prior l o Wealment with meuonidawle. In convast prior meuonidawle treatment significantly decreased the ability o f the PCR to detect E. hbfolyna DNA in the liver abscess pus, with only 53 (70.6%) of 75 liver pus samples positive (Fisher's Euct test, P - 0.0006). The probability of E harotylua DNA detection in liver abscess pus by

i i l ( ~ I % ) of 37 liver a b x m pus o f paticnu which were nested multiplex PCR was 31 times more i n patienu who ~c~llrctcd onor to treatment with metronidawle In con- had not rercived ~ r i o r metronidawle theraw (OR - Ir,,rt. the Sechlab E hutolpc-a test detected the lenin anti- wn In only 26 (25.5%) of 102 liver pus (XI- 32.61. P c ' I 001 ), collected aher initiation o f therapy n i l h metn~ni- drtulr Ihe probability o f E. hisrolynw antigen detection II Itvrr abscess p u s by €USA was found to be 12 times nlc?rr In patients who had not rrceived prior treatment wth mnronidamk(Od& Rado (OR) - 12.53. 95% Con- fidrn<e Interval (Cl) - 4.55 m 35.86) than in the patients *IIO rrceind prior mcvonidawle therapy. l h e OR was ~trt~st~cally significantuthe 95% CI ofOR was more than

\llmnropy o f he liver pus demonstrated E. h~~tolyltw Wo- Vhrm>ltes i n 10 of 139 (7.2 %) liver ab~ess specimens. hul only 2 (1.4%) pusspimcns wmpositive by nllture 611 1 hrrldynco. All 10 paticnu w h w liver pus were pas- lllrc for t, huwlyriw by microscopy and/or culture were

posit~ve for E. hmdpcn Gal/CaINAc lertin antigen in be llvcr pus by he Techlab E. hirtolyncn II test and serum "Ilwbic antibodies by the IHA test.

f lolal of 102 out of 139 (73.4%) liver abxas pus were nryltlve for aerobic bacteria by Cram's staining and bac- tfual rultun. 'lbmcy rm liver abscess pus specimens WPrr positive for aerobic bacteria by Cram's staining and

. . . 11.54, 95%CI - 1.879 to 624.2) than i n the patients who received prior metronidawle therapy. The OR was sfatis- tically dgndcant as the 95% CI o f OR was greater than 1

'lhe nested multiplex PCR did not detect E. hwlgncd DNA in a total of 49 liver abxess pus specimens, which included 27 PIA and 22 AL4 pus specrmens. The proba- bilrty o f negalive nested multiplex PCR results, in these 43 liver abscess pus specimens due to PCR inhibiton was ruled out by the induslon of an internal amplification conlrol (IAC) in the nested PCR reaction.

'lhe comparison of results o f nested multiplex PCR and Sechbb E. hiswlyncn L I EUSA test on liver abxess pus hom MA patients is r u m m a r i d i n the table I

The nested multiplex PCR m u l t on urine specimen is shown i n Figurc I. The nested multiplex F'CR was performed on urine specimen collecled fmm 68 patients (including 53 ALA and 15 PIA) and 43 conuols. The nested mul t ip la PCR t a t dctmed E. hirlolyfiw DNA in 2 L (39.6%) o f 53 u r im samples collffled horn patients with MA (Table 2). The test did not detm E. hiswlyricn DNA in urine samples collccced from all 15 PIA patients and 43 controls.

O L h- w - by mkrnrcw rider mkwc m 10 of h e 55 W md K R Inn lbrccrr pus specimens.

'lho nested multiplex PCR test detected E. huwlytica DNA in the urine specimens of 4 (17.4%) of 23 ALA patients who were tested prior to treatment with metronidawle and i n 17 (56.7%) of 30 ALA patients who were tested aim treatment with metronidawle (x' - 6.83. P = 0.009). ill of the 4 ALA patients, who did not receive prior treatment with metronidawle and whose urine specimens wore positive for E. hiswiyhu~ DNA. were available for follow-up ILudy. Urine specimens were collected from these patienu every week for 4 weeks after starting of the ther- IPY with meuon~dawk; and were tested for E. hlcrnlynco

Mrcuuion In this study, we have made an attempt to detect excretory E. huwlyticrr DNA in urine by applying nested multiplex PCR and to assess the diagnostic potential of the test for detection of E. hisrolytica DNA i n urine for the diagnosis of ALA. Also we have studied the kinetics of the excretion o f E. hiswlytico DNA i n urine during the course of therapy with meuonidmle.

In our study. 76.8% (86 o f 112) o f ALA patients were pol- itivr for antiamoebic antibody i n WNm by the IHA. This

DNA by PCR. It was o k r v e d that 2 wccb aher treatment result was similar to that reported horn Bangladesh where ~ i l h metroniduole. 3 (75%) out of 4 urine swcimens %rum antiamoebic antibodies were found in 78% of ALA . , hprame nmt ive for E. huwlyfrc. DNA. One urine spcci- patients 1101, but differed from that of the study reponed men k a m e negative for E. hrstolyhca DNA aher 4 weeks from South Africa, where serum antiamoebic antibodies ui ~rratmcnt with metronidamle. were found i n a higher 99% of AIA patients (351.

Only two out of 10 ALA pus samples which were positive for E. hlrrolyhui trophowite by m ic ro~opy were positive for the amoebae by culture and the rest were negative, this may be due to the inhibition ofg~owth hy culture itself. In maiority of patients. K. pneunumim was the mapr bane- nal pathogen responsible for PLA and as secondary bacte- rid infection of AIA. One of 10 NA pus specimens was positive for Croup B .%lmarlla species, thls patient had l imabsces with perihepatic collection, with severe sepsis and disseminated invavascular coagulation, finally the patient died. I n this study. the anaerobic culture of liver abscess pus aspirate was not done. Therefore, the possible ctiology of liver abscess due to anaerobic ownisms such M Biacrnoida remained undetermined.

In the present study. 50% (56 of 112) of liver abscess pus w m positive for E hlsrolyaca lectin antigen. 'The sensitivity of the teat i n our study was observcd to be slightly hiia than that of the study using the same Techlab E

Reult d md mukpkx PCR on representative lim hr;;dytlccl II kit (40.7 % sensitiiWy) on liver pus reponed

pn =,,,, urhc *, hiam (EH) h m Bangladesh 1101. However. reaulls o f other studies

maru~ rnp~~~w c-al (IAC) ur 439 305 U"i"B ~ o l ~ c l o n a l antibody based EUSA *owed a very ap r*, bne-l nd 4 v~ E high sensitivily for the de+ectionofamoebic antigen i n the DNA h ~m k m M -m m e , k p. Amoebic antigen was detected i n liver absceas ~ ~ ~ . l n d ~ ~ ~ f a ~ ~ D t & b ~ ~ . p1n976%(41/42)ofALAcwsbyEUSAasrepoRcd I w bp DNA W Imei. w) horn Ch~na 1211 and i n 92% and 96% of llver pus by

U A . 53 ,N.2i . . , . . .

wv ln 29 (9 n o i113r.G ' o " ' 6 RI. I S 0 0 0 0 0 0 0 0

. -... - . . - . . -- - - .. -- -- ~mocbr her absceu: Wptc h m *cur

uslng immunoclenmphoresis and EUSA respectively. lrom India 1361.

In developing countries like India where amoebiasis is mdcmic. antiamoebic d r u g and antibiotics are used ~odlscriminately, making i t difficult to obtain an accurate treatment history. Most o f the patients in the present rludy had already bee11 treated wrth meuonidawle at the itme of collection o f cl in~cal specimens. The serum amoe. b ~ r ant~bodies were detected i n hinher mrcenmee 194.7%) - . v . . oi .AIA patlents treated a r l ~ e r w i th metroniduole, but sere dc tmed i n only 40.5% o f patients who d id not rrwlvr any prior treatment with metronidawle. This might be due to the law rnubody responx during the ruursc of the disease

Ilnilkr serum amocblc anubody detection. E , h u u l ~ n u Ikcltn antlgen was detected I n l ~ v c r pus byTcchhb ELJSA ~II a l ~ r ~ h e r percentage (81%) o f A M patients, who were Icrlcd pnor l o treatment with metronidawle, but was .J<tcctrd an only 34 6% of A lA patienu, who were tested I lar the tnidatron o f therapy with metronidazole lh is n~lgltt be due to the rap~d clearing o f amoebic antigen Imm the liver pus due to ki l l ing of E. hu idpca tropho- rolt1-3 o n trraunrnt w i th me~ronidazole.

Ihc I'CR for the detccaon o f E, huiolpca DNA in liver 1buc3s p a had a much htgher semitivity (100%) when l~ isd pnor t o treatment wlth mtuonidwole. but had a

1 iclwer sensitivity (70.6%) when t m e d aher the inluation ftrratrnent w i th mctmidazok. ' Ih is might be attributed 10 lhc clearing o f E. huidytlcn DNA f m m the liver abscess dllr 10 the death and 1y.L o f E. hutolynca tmphozoi ta o n t"ntmmt wi th meuonidazole ?he pcmenuge of agrec- nlml bctwem E , huiolytlcn DNA detection and WS.4 for t huwlylira antigen dc tm ion in lim abacus pus was Clund to be 67.9% i n thc p m c n t wdy (Table I ) . The

statistic was found l o be 0.36 which indicates a fair 4Klrnnent between the two tests.

PCR for detection o f E. hiswiyt~cn DNA and EUSA for detection o f E. hirwlytira lectin antigen in liver abscess pus were evaluated for the diagnosis o f AIA (McNemar's w' = 30.25, p < 0.0001).'Ihe sensitivity o f PCR was 80.4%. This was found to be significantly higher than that o f EUSA (50 %) w i n g McNemar's test (p < 0.0001). A l l 27 liver abscess pus aspirates diagnosed as PIA were negative for E. huwlynca DNA by PCR and for E. hawlytun l d n antigen b y T m h h b EUSk which represents a specifidy of 100%. EUSA for detection o f liver abscess pus E. hiswlynca lectin antigen demonstrated a 10096 positive predictive value and a 32.5% n w t i v e predictive value. PCR for the detection o f liver abscess pus E. hutolprw DNA demonstrated a 100% positive predictive value and a 55.1% negative predictrve value.

In the present study E. huulIyocn DNA was detected in the urine specimen of 4 (17.4%) o f 23 AIA patients, who were tested prior t o treatment wlth metronidawle and in 17 (56.7%) o f 30 AIA patients, who were tested aher treatment w ~ t h meuonidamle by PCR. l h e probability o f E. h u i o l p ~ w DNA detection i n urine by PCR was 6 times more in MA patients who had received prior metmnida- role therapy (OR - 6.2. 95% C I - 1.47 to 28.37) than i n the AIA patients who d id not receive prior metroniduole therapy. The OR was statiaically significant as the 95% CI o f OR was greater than I. This might be due t o release of increased F , htsrobnn~ DNA from the dying E. hiswlyt~ur trophowites when metronidarole therapy was initiated. leading to acret ion o f E. huwlynca DNA in the urine. One study has demonsuated that the DNA from dyingcells can c rou the kidney barrier i n rodents and humans and can get excreted wi th urine, which can be used for genetic analysis 13 11.

PCR for detection o f E. h u i o l p u I>NA in liver abscas pus and urine specimen were walualed for thc diagnosis of A U (McNemar4s ~ 1 - 26.28, p c 0.0001). 'lhe sensitivily o f PCR for urine was 39.6%. 'lhs was found to be signifi-

Page S o l 10 ( p 9 . " " m . , " d b r a m m

randy lower than that o f PCR for liver abscess pus (80.4%) using McNemafs test ( p < 0.0001). Al l urine specimens from 15 PLA patients and 43 control group ~ndlviduala were negative for E. huwlync. DNA by PCR. lhir represents a specifidly o f I O N (Table 2). PCR for (he detection o f urinary E. hiswlytiur DNA demonstrated a I 00% positive predictive value and a 31 .% negative pre- dlrllve value.

.\r per the PCR k ine i ia the likelihood o f ampl~fy ing smaller PCR producc is more than amplifying larger PCR Ip~duct We feel that i f lhe PCR product smaller than 400 hp would have been amplified. the PCR might show li~plier sensitivity.

I h~rrolprrd DNA i n unne did not persist longer i n A U patients aher treatment with me t ron idw le as observed III the procnt audy. Three o f 4 (75%) urine spermens I~>,IIIVC fort.. hicwlynm DNA became negauve for E. hu- a111~11rd DNA within 2 weelu o f treatment with metronida- u,lr This mipht be atuibuad to the reduced acret ion o f I h ~ ~ r ~ r u r DNA i n the urine as a r m l t o f reduction o f E. Ihc~c,l>?lra i n f ~ t i o n load follow in^ treatment wi th metro. ~ ~ ~ d a r o l e 'The effect o f metronida7ale i n ki l l ing o f E hlcro. li,frt~ arid c l ea r i n~ o f antignemia i n hamsters suffrnng lrum hepatrc amocbrasis has hcen well documented i n a rludy reported by Ihammapalerd a al. (371 Results o f the present study therefore indicate that the PCR can he used

monitor exr r t ron o f I<. hutolprur DNA i n urtne as a I'IO~IIOS~IC marker d u n n ~ therapy o f A IA wrth spfcrfic ~ l l l l d m ~ ~ b ~ c dm@.

III ~l ir prrsent study. none nf the liver absrrrr pus and untw PCR resulu were posrtrve for esther E dlsplr or E. ri~ahkonkr~ specifir PCR producu, whlch confirm the nor1 IIIV.*\IVC r lr turr o f these species (l'ablc 2)

its drtenion of E. hlcmlptnl DNA and E. h a w l y r ~ ~ a spt.. ,1111 lkctin antigen i n the W N m speclrnen o f A I A pativrits *,IS not carried out i n this study A controlled prospcctlve 5:edv to evaluate thc d e m i o n o f E. huf04~1u1 DNA and h 'ifia~l).nrrr s p r t i f ~ kc l i n a n l i g n in the serum specin~en of

nattenu has been intended to be carried out I n future

Conclusion lltr Irrcunt study for h e first time s h o w that the kidney h~rrlrr i n AU patienu is p m a b l e to E , hutttlyhra DNA ln~olrruk resulting i n acret ion o f E histolyncrr DNA i n urlnc which can be detmed bv PCR. 'lhe Uudv also s h o w 'hrl the PCR for detcction o f E , huwlyttur DNA i n urine o f

urine specimen o f AU paticnu prwides a new approach for the diagnosis o f Alh.

Methods Sow dudh ?he subjects in the present study included 139 patients orovisionallv d i a m o v d as A U . who were admitted t o , .. IIPMER hospital. Puducheny, as well as 43 contmls during a period from September 2004 t o March 2006. The provisional diagnosis o f ALA was made by the physicians o n the basis o f patient's history and clinical features, unfortunately these features are ohen nonspecific to confirm the diagnosis o f ALA. O f the 139 provisionally diagnosed AU patients, 102 had received prior treatment and 37 d id not receive prior treatment wi th metronidawle. The patients and controls were residing i n neighboring area o f Puducheny, Informed consent was obtained from the paticno. This study has been performed with the approval o f Institute Research Council o f l lPMER Puducherry.

The control group included 43 individuals who had n o history o f recent dysentery or dianhea and whose stool samples were negstive for E. hiswlynur infection by microwopy and culture. Thirty five o f the controls were healthy asyrnptomatrc volunteers. and the other 8 patients included, hvdatid cw in l i v n ( n - 2), liver hevatoma ln . . - I ) , liver cirrhosis ( n = 3), and viral hepatitis ( n - 2)

The diagnosts o f MA was established o n the basis o f radi- ological. symptomatological and laboratory criteria as fol- l o w 13.101. ( i ) a space-occupying k r i o n in the liver diagnosed by ultra sonography and s u w t i v e o f abscess. ( ~ i ) cl in~cal symptoms (fever, pain i n the right hypochon- drium (often referred to the epigastrium), lower chest. back o r t ip o f the nght shoulder). ( i i i ) e n l w e d and/or tender liver, usually without jaundice, (iv) raised right dome of the diaphragm o n chest radiograph, (v) improve- ment aher treatment wi th antiamoebic dm@ (e.g.. metronidazole) (vi) positive IHA o f serum antibody showing a tiler (2 1,128) against E. hisu)Iyrh, (vi i ) liver aspirate appeared like anchovy sauce but was bacteriologically sterile.

-p*- bver abscess p: The aspiration o f liver abscess pus was indicated only under the following conditions 131. (i) t o rule out a pyogenic abscess: ( i i ) the failure to respond clinically i n 3 to 5 days; ( i i i ) the threat o f imminent mprure; and ( iv ) the prevention o f m p t u n o f leh-lobe abscess in to the pericardium. The liw abscess pus aspirates were per-

Pnll~nu wi th AIA can also bc used a; a prognostic maker f o m d . only for clinical purpmes as tud& by the clini- '0 nssenr h e coune of the diseases followins theraov bv cians for the ~a t i en r care and not for the nurwse of this .. . , , . . - nroniduolc. lhc d n m i o n o f E hutobzan DNA I n study. l i ver abscess pus was obtained under ultrasound

guidance from all I 3 9 provisionally diagnored A U

pattents and waa stored at -20'C i n a sterile container *ntlcmoeblc broprd.iHA tnt untzl 4. The Rapid-IHA was pnformed on serum specimen as per

the mnhod described earlier 1391. Briefly. double alde- lnne Urine spcclmcn could be collected hom 68 out of hyde strb~lized chick red blood cell ( ~ ~ ~ ) s m s i t k d with I 3.1 pronriondly diagnoud AIA patients and a11 43 con- the ammbic anupen was uud In the test The haemadu- trol grwp individuals included jn the nudy. l o ml of urine specimen was collected in a sterile container using nrptic techniques; urine sample was stored at -20'C until "U'

u l d : Blood specimen was collected from a11 139 provi. stonally dirgnoscd AlA patients and 43 control group individuals included in the study. Venws blood (5 ml) was rollmed in a sterile containec sera were separated a~ld stored at -20'C until used.

Micmlcopl for EnLunocb. liver abxm pus: 7he specitnew were aam~ned immedi. rlov dfier IIK aspirauon of abscess. lhe liver absceu pus uar first centlifugd at 2.500 g for 5 miw and a loopful usually inomlatinu needle loop) of sediment was mixed . . n t h a drop of warm saline on a microscope slide Micro. rkoplr examination of an amoebir abscess aspirate hom !wcr may meal haematophagous tmphow~tes

C " u r r for Entunocb. lirrr a h e m INN l~ver ahxtss pus slwcinicns were cul . . :urrd lor ~ n & w b a spccies in 1ncke.m (If) medium \Ill modtficauon of Rocrk and ~rbohlav's medlum) as ,rw~oudv dcscnbcd 1381 Ihe liver abscess w s was Crst xntnfu& at 2,500 h fo'r 5 mlns and a lodpful of dl. tlmt w;s ~nomlated mto the lE medium It I; to be noted :hnl In raw of culturim t'nrnmabo from Ilm i b x w asna- '~Icx, sinre thc abve.s IS sterile, barterial flora (AI'C:C F

'!I] war added hefore tn-lat~or~ of amolrhae Into xenv ililllrr

Gmm noin ad boctalaf e u h for IIVU obsceu pus ospnrmcr l'lrrrt smear (,ram stalnlng and bactenal culture was 41 111 for all Ilver a k a pus asplraw Ihe her abscess Vu% sinutmuns were ~noculated on to sheep blood agar 'la(x,nky agar and chocolate agar plain Ihe M A O - "n AK." plates were incubated at 37'C for 18-24 houn *lxans th. blood awr and chocolate aer plate were '"(abated ~n a candle jar at 17'C for 48 houn Based on l'le ll)lor~y morphology and r u l t of culture smears nec- ' W v biahcm~cal t r w were done to tdrnufy hmena to t'l? SWCl'3 lml

I ~ h l o b E. 11 ELISA m r lllr Iechbb E. h r u ~ b ~ a II test was prformed on liver "hum pus specimens to detm E. hirdynu ar per the "flIl(xl dercrlbcd carlier 1101.

- tinalion test waa pnformed on lest serum samples including known positwe and negative control sera in each batch.% chick RBC yttled quickly and their haerrugglu- tination pattern could be determined within 30 to 45 min of incubation at room temperature with test sera 1391. A titer of 2 1: 128 was considered positive for AlA 1401.

htmcuon of Enbmocbr pnomk ONA Liver abxess pus: Rriefly, 1 ml of liver abscens pus was taken in 1.5 ml centrifuge tube and centrifuged at 12.000 g for 8 minutes. The supernatant was discarded and 50 pl of pellet was dispersed in 250 p1 of lysis buffer (0.25% sodium dodecyl sulfate (SDS) in 0.1 M EDT4 pH 8.0), followed by addition of 100 pdml of proteinase K. The lysate was incubated at 55 'C for 2 houn. Ihen 75 pl of 3.5 M sodium chloride (NaCI) followed by 42 p1 of 10% cclyltrimethylammonium bromide (CTAB)/0.7 M NaCl (heated to 55 'C) was added. After the components were mixed. the sam~le was incubated at 65'C for 30 min. This was followed by enaniow with equal volumes of chloroform and then phenol-chloroform-imamyl alcohol. and the DNA was precipitated with ice cold e~hanol. The dried DNA pellet was dissolved in 50 p l of sterile distilled water.

Urine: Bnefly. 10 ml of h e urine sample was centrifuged at 12,000 g for 15 min at 4 .C The supernatant was discarded and the pellet was suspended in 500 p l of sterile distilled water. The suspension was boiled for 10 minutes followed by sudden cooling. Next. 5 &I of proternase-K ( 10 mdml) and 60 p1 of 10% SDS were added to the sus- wnsion and incubated at 65 'C for three hours. Then 80 ;I of 5 M NaCl and 20 pl of 10% CTAB were added to the mixture and incubated at 65 .C for 45 min. This was followed by extractions with equal volumes of chloroform and then phenol&loroform-isoamyl alcohol The DNA was preciditated with icecold ethanol Thedncd DNA pellet was dissolved in 50 pl of sterile distilled water

The protocol for enraction of DNA €ram liver abscess pus and urine specimen has been modified in our lahonrory from CrAB DNA enraction protocol originally described for DNA emaction fmm amoebic culture 1411. The atracted DNA hom liwr abveu pus and urine sample was pawed through DNA clan-up spin columns (Rangi- lore Gcnei KT-62. Bangalore). The DNA was uored at -20 'C until uud.

puot@cUon of DM4 In Ih.r obxcas pus ond vlm ~pecrmn VNA quantification in spin column purified DNA extract hom liver abscess pus and urine specimen was deter- m~ned by UV abmrbance using a Cintra 5 double beam spucvophotorneter. DNA yields were calculated on the h.tris of IN absorbance x dilution. The puriry of the nucleic acid in the samples was estimated by the ratio of readings at 260 nrn and 280 nm (ODIJOD,,,).

m- krll" Ihc genus speafic primers wne deigned using nucleotide stquences of 16Sl ik rRNA gene of E. dispar, E. hisro&irn and E. mmhlrwrlrri Laredo deposited in GcnBank lacces- slon number: iWU.561, [accession number : a 1 and /ncession number. -1 respectively.

Ihe comparison of all the three IGSlike rRNA gene rcquences of E. dupar. E. hrrurlylun and E. rnoshkovrkii larcdo revealed significant differences enough to design ywcies specific primers. In this study, we have used an 14L targeting human I8S ribosomal RNAgene to rule out lrlw-negative results in clinical specimens. All the primerr \*'ere designed using Prime3 online wfhvare 1421 'Ihe prlmers used in PCR an shown in'rable 3.

Standard stmlns lhrrc standard strains ured in this study were E hurolyrun tI1i.1 IMSS. E , dupor SAW7GO. and E. rnoshko&i laredo Ihne wtye used as @live control in the present nudy. Ihr lvophilized DNA of these $trains was generously alt1t.d by Dr. C. Graham Clark from lnndon School of !Irg~rne & l'ropirrl Mcdicinr. London, 1IK.

Nested muhlpkx PCR powcol Liver abscess pus PCR: For a reaction volume of 25 yl, comprising 2.5 pl of 10X PCR buffer (Biogene), 2.0 p1 of 25 mM MgCI2 (Bangalore genei). 0.75 pl ofdeoxynuc1eo- side triphosphate mix ( I 0 mM each d m , Biogene), 0.3 pI(5 IUjpl) of Tq polymerase (Biogene), 10 picomoles of target DNA primers (IDT) and 5 picornola ofIAC primers (IDT) were added in genus and species specific PCR. 'he template DNAvolume was 2 p1 for bothgenus and species specific PCR. The PCR tubes were finally placed in an EppendorfThermal cycler [Master cyder gradientl.

Urine PCR: The PCR mix composition was the same as described earlier for liver absceu pus PCR except that 1.0 pI of 25 IIIM MgCI, and 2.5 pl of template DNA was added.

The conditions for genus specific PCR were as follows; the PCR mix was subjected m an initial denaturation at 96 'C for 2 minutes, followed by 30 cycles - each consisting of 92 'C for 60 seconds (Denaturationl, 56 "C for 60 seconds (Annealing). and 72 'C for 90 &conds (Enension). Finallv one cvcle of enension at 72 'C for w e n minutes , , was performed. In the species specific nmted multiplex PCR (which had multiple primer see in the same tube). only the annealing temperature was changed to 48 'C, leaving the other parameters of the amplification cycles unchanged

3.5 p1 of the amplification product was separated by elec- trophoresis through 1.8% agarose gel (Agarose Lmv EEO, Rangalore genie produns. Rangalore. India) containing ethidium bromide in 0.5 x Tris-borate-EDTA at 120 V for 45 min and was visualized under UV light for bands of DNA of appropriate sixs (Figure 1) A negative control

Gmus spohr pnmo ( F m PCR) - . - - . -

E- 1 5 TAAGATGCACGAGAGCGUA T (hnnrd pnmr) E.2 5 GTACMAGGGCAGGGACGTA I' (m-sc pnmtr)

- -- -- -- -- - Spec- rpc~f ic p n m m (Second n e r d m u l w K R )

- - - - - - -- - - - L - W En- l 5 MGCATTGTTTCTAGATCTGA I' (- -)

EH.1 I' M G A G G T C T M C C W T T A G 3 (men* prvna) L e l p c c k r 1 6 s I I' G U A C C M G A G l T K A C U C 3' ((Mud -r)

flas.15' CMTATMGGCTTGGATGAT 3 (nnn. mmr\

l n n d mpAk.lon c a t m l (LAC) pnmn & K R

Page 8 of l o ~ N m r n * ~ s k l s n ~ I ~

rcaction waa included with each batch of samph am- lr,ed by PCR.

p r i m wndmfall Ihe primer sequences designed for E. moshkmkii, E. his@ hord. E, mspllr and Human IAC were subjected w Basic I c~a l Alignment SearchTool (BLAST) in thegnome database of all organism 1431 and were found to be specific for the study. The amplified FCR produnr of E, hrrwfytica species in liver abscess pus and urine samples was con- lirrned by getting both the strands of DNA sequenced on 4H13730XL sequencm (Macrogen. Seoul, South Korea). Ihe sequencing waa done using species specific primen I c EH-I/EH-2 for E. hirwlytica. All rwqumces were ana- lned for homology by using the nucleotide-nucleotide lilAS search feature 143).

Ihe identity between the sequencing results of PCR prod- url of E. hurolyaur fmm liver abxes pus and urine with thc sequence deposited in GcnBank [accession number: Si62211 were anal+ by using the 'Align two wquences (hl2sq)' feature 1431.

lssenmwn ofcmnpuhbn ofnon toqet DNA [luring thr standardization to a w u the competition of ullwr non target DNA present in liver abscess pus and urlnr specimen with ta@ DNA. the nested multiplex In( H was checked with reference DNA (DNA from stand- J I ~ culture of E. hurdyr~ra, E dirpar &:. mhknnbt~) splked with DNA from liver ahsres pus (PIA pus negallve Ihr I , hisrolytiro) and urine (negative control group) fol- ILlwrd by nmed multipla PCR amplification.

L*nwtloa of mlnlaum m m k of Encunocbr celk dttcMbk by mmd mdrlphx PUI Iliir wds performed for E hurdyficu with Locke-egg (1.E) scll~um (Nltl modification of b e c k and Drbohlav's nirclium) liver abscess pus cultures: the amoebae were ,i,unted using a standard haemocytometer. A cell pellet containing 106 cells was preferred for determin~ng the drtrction limit of nested multiplex PCR for E harolyrtn~. !be cell pellet containing 106 cells of E. hisrolynw was dliutnl 10 foldt in phosphate huffer saline (PIS) to l~htain different concentration of cells, such w lo5. 10'. "1'. 102 a d 10 cclls/ml. 'Ihc different dilutions of cells l,111g1ng from 10h to 10 ccllrlml were centrifuged and the 'pnl~~ning pelln of each dilution was added lo 0 05 ~1 of lfi'et I ~ S F C ( U pua (PIA pus negative for E. hismlyncn) fol. 'owt'd by DNA extraction and PCR as pcr the aforemen- ll"l~rd protocol.

~~d data emlph k:ls~tivity wm calculated as followa: number of patients 'Ith positive ~ l t rrsulcs/total number of paticnu x 100 'l~rrlficiry was calculated as follows: number of controls

with negative test results/total number of controls = 100. Ihe positive predictive value was calculated as follows: number of true positives/(number of m e positives + number of false posidm) x 100. The negative predictive value was calculated as follows: number of uue negatives/ (number of m e negatives t number of false negatives) x

100. ']he agreement W e e n the tescs was calculated using the Kappa statistics. To determine the statistical significance of differences between the proponions, Chi-square ( x 2 ) test and Fisher's aad test were used. The X' test and odds ratio were found using 'Epi Info Version 6'. To cal- culate the significance of the difference in sensitivities, McNemar's Chi-sguare test was applied. ']he McNemar's test was performed using 'Graph Pad Software'.

Authors' contributions SCP s u p e ~ d and coordinated the study, and helped to drah the manuscript. KK camed out the experimental works, and drafted the manuscript.

Aclcnowledgementr We r*lr& dm& Dr. K.V. I0rubwhr a d Dr Gow-hr horn

References I WaM Heakh Orgmzwm *mOcaurr Wm) W w Epdrmd

1- 1597 7267 IW 2 SmqnWP*dr(lCK,P*QIeSkPyolmuYrrrrhsrcuerr

compvl-doldumd-p.ana &~AI.~IWB. 17443 448

3 Gspr DL %rmdd L huo AS k u u r Y. Lonp DL J a m a m JL H a m a s p ~ # n r m o l r m d a 16m- US*.McGnw Hdlc~mp~IrcZaKl l l l l l lb

4 K a m n x e m D kk- V &wde New rmccpLl d unebk 11- abxnr d c d brm hepatic imqvn& ~ r a & l n a i s and h e p a e m p e a n 67 ~ o m u D v e cucr n 5.n Mego hWwe IbduweI l98l 61:23746

5 G n d h i ~ h ( u d M C M I T C T u d m B N C n m m l m M ~ cein *: an E U Y kr &Bstion 01 amoebic &tibody. R k TropMcdw l987.81:183.11.

6. Jackson TF. Gm*m V. %qee AE: Sw(olW dMsmndatia beween pst .nd parent Inkctia In hcpltic mb*rik. rmm R k Tmp Mcdm 1981.7L341-345.

7. Y a y J. K m w M. E n k u h d e k d .dm- rabentvvrk rda=em&mc&d~octh& IC!+Mm aa, 1vn. ~s i ra lss RnpSC I l t r m t o a n d . h h s ~ d ~ b i ~ 1 1 ) k ~ psUm M o 1991.41:Ul3-385

9 Z l m u l ~ K h m / ~ S W . U m e d R K h a n M h k n R t m n V D h c t mpllllutb, d Emmmeba Mud@ca DNA brm .mmbkUrrr .Lurp l ru~po)*muchin~. h d R" lmo. 8b:M-e

10 Hacehr(drhNU.AYI&AbmK.-hL&D,Rm

Page 9 of 10 (p.g.NnbrnXhvoOmnluPwt

11 2mh. G h d m R N~hma*b 'I. U*ra-l w. Pkdmn D

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Your research pap^ will b e . .dl.b* hl d mYOc to the nave bbmdiid comnunlh

Page 10 d 10 ( L v p M l b r r m b n r U m W S l

Diagnosis of intestinal amoebiasis by using nested polymerase chain reaction-restriction fragment length polymorphism assay ~KISIINA Kltnla~aa. SIIRIIASII ('IIANIIRA PARIJA, and RAVISANKAR PALANIAPPAN

!hl~drtrncnl of M~rohology. hwaharlal lna~luk d It*itgnduute Mcd~cal Education and Rcrarch, Pondicherry 605Mh. Idin

R a r k p w # d Micrnscopy is unreliahlc to distin~uish the pr~ht~gcnic b.'n:nmmoeho hisrr~lyrrt.~~ from the nonpatho- rcnlc b.nrontcwbu dirprrr or Ettrrrn~oehu n~orhkovskri 11 \IIU~I rpecimen~ Medodr Nested p~lymerax chain ;..tct~(~n-rcartction fragment length polymc~rphism IY'K-RF1.P) was carrled nut to dctcct E hrvrr~l~rire. E. ,li,l~~r. and b:. morhk~,rskrr DNA in stlx)l \amplo of 202 p.~tIcnth p~silive k t 1.:. I~rrrolyrrrrr. E drrpur. or F rnosh. i t~skii hy mrrtmopy or culture and In 35 contrc*l~Ihc l ,~l~l . ;~h t., hrm~lvncrr I1 cwymc-linked mhnuntnor- b:nt :Itsay (ELISA) was pcrformcd detect GaV !~rlNAc lectin In 45 s t c ~ ~ l samples pcnitive for C:. hhro- ' t : ~ ~ o . I: dicpur. or I:, n~o$hkfrurkrr by mlcrowopy or .sllulc Rapid-indirect hemagglutination a ~ u y (IHA) 'Arr performed to dclcct scrum antiamcrhr antlhdles n the XS patients paitive fnr E. hrsu~lyrrrrr. I: dbprrr. ! I nr~uhkovrkit In the~r rtcx~l rpcclmcns and 111 the 35 ,lnlr~~l\ R n v l u Nsstcd IY 'K-KFLI' was p~s~t ivc in '< ot 202 (%.6%) patient r t n ~ ~ l s~mples and was ncga- ',li In all 35 negatlve cnntrnl st~nll simple% kl.lSA war '\8\lllrc tn 29 of 45 (M.4'%) patlent sltx~l \amplc\Ihc :!I,\ ILN was pc~ttlvc In 10 i ~ f 85 (22.4'L) patlcnt rcrum !!1111lcs and in one (2.8%) of the 35 control scrum ,~mplca Ncstrd P('K-RFLI' dctectcd t. hrsrolvriccr I'Y,\ ~n stcr,l spcctmens of I2 (63.2%) of 1') wruposi- 1 8 ~ pdlienL\and in 31 (47%)0f Mxronegattvc patien& 1:~hl.ilh I:, hu~o /~ rca I1 ELISA detected C hrsrr~lyricn ~ntlsan In s t n ~ ~ l specimens of hix (54.5%) of 11 rrop)si- ! ' L p;lticnt& and In 23 (67.6%) of W wrnncgativc :allmtr Corclurlon+ Ncrtcd ll('K-KF1.P was usclul 1 1 Ihr \pec~fic detectk~n of I: h~vrr,/yrrcl?. t drspur. and I ~ ~ l ~ ~ ~ h k r ~ v r k r i in SICKII samplea

b! wwdr 1Y.K. KF1.H I... n~r~.rhk~rv.skrr. Puducherry. Ind~;,

hcl~,vcd. April 13. XXI7 1 Accept~& May >I. XX17 k , ' ~ ~ l ~ ~ ~ mqurtllrn: s.(.. Panja

Introduction

Enrun~orho hirrolyricu causes amoebiasis including both intestinal and extraintestinal amoebiask Ent- amoeba hi.~rolydcu results in 34 million to 50 million symptomatic caws of amoehiasis worldwide every year. causing 4)W to I(X)O(K) deaths annually.' E;nuunwhu hhrolyrica. the pathogenic species of amoeba. is indis- tinguishahle in its cyst and trophozoite stages from those of E. ntoshkovskii, a free-living amoeba,' or E. disper, a noninvasive amoeha. hy microscopy, except in cases of invazive disease. when the E hisrolyricu tropho- mite may contain ingested red h l d cells' hut such a finding is rarely seen.'lhis leads to a confusing scenario for the definite idcntification and differentiation of E hr.rro1vrrcu from E mnshkovskii and E. clispor in the diagnosis of intestinal amoehiasis'

I hc sensitivity of light microscopy is limited to only W% at best.'" Istrrwyme analysis of cultured amoebas allows the ditfcrcnliation nf L hrsrolvricu from E. drspur.' An iscwnzyme analysis taka usually nne to scveral weeks kfore the resub are reported. and also requires spcchl laboratory facilities making it impractical for usc in the n~utine diagnosis of intesdnal amoc- hiass Moreover. factors such as delays In the stool specimen prnccssing and initiation of antiamwhic treatment prior to sample collection can lead to E hisrolyricu-negative culture results even in those patient s t a ~ l samples showing cysls/trnphozoites hy light msrusccvy.

At present. ammbic coprc~antigen detection and p lymerar chain reaction (PCR) analysn of stool samples arc rep)rted t o k specificand wnsitivc methnds for detection of E. hisrolvricn in stool specimensu l1 Ibt. sensitivity and specificity 01 monoclonal antihdy-based enzyme-linked immunosorbent &\3y (ELISA) for the detection of I<. hisio(vhca-specifie antigen5 in [he stool specimen ol intestinal ammbiasis patients have k e n rep,rted to be KS% and W%,respc~tivclv." ELISA for

K. Khaimar el al.: Nested PCH-KFLP for 1;nramoebu

antigen detection and nested PVK targeting the 16s- like rRNA gene of E. hisrolyrica have shown compara- ble sensitivities of 85% and 87%. respectively. when Frlormed dircctly on fresh stool specimens.'"lhe main i~m~lation of ELISA is that it can specifically detect I. t~isrolyrica and E dirpar but not E, moshkovskii.

lill now. E. nroshkuvskii in humans has h e n rcp)rted North America. Italy. South Africa. Rangladesh.

;and India.'," Our report of E, moshkovskii was the first ,uch rrplrt f n ~ m India."lhe presencx of morphologi- ,:lily similar k ) m s such as E dispar and E rni>.ihkorrk~r h.15 made the differentiat~on of the three species hy IY'H necessary, especially in those countries. including InJla. where all three species E hisrulyrica. E. ilispar. ,~nd f.'. nroshkowkii, have been repjrted to hc prcvalcnt.

Ihc differential diagnmis of pathogenic E, hisrolyrrco tram ncmpathogenic E dispar and free-living E. mosh- ilor\kii will enable clinicians to avoid unnecessary Ircat- lllrnt with antiamcxbic drugs, as coloni7;ltion with E .il,pnr and I.. . rnt~.~lrkovskii does not warrant antiamcn- b~c therap!

\ total of 1720 slcu)l samples were collected and cxam- ~nrd from paticnts prescntlng at Jawaherlal Institute .>I lhstpraduatc Medkal Education and Kcrarch III'MEK) hnspltal. I'uducherry. India, with general

~u~mpla~ntr of gastrointestinal drscomfort durlng July !I#U to July ZOOh. As negative a ~ n t r o b in thc prcscnt \iudy. we uwd 35 rtcx)l sunples collcctcd from healthy ;xcru~ns a\ wcll a\ patlents with other intestinal infec- 'I'm\ who \ h ~ ~ w c d no trophon~iter or cysts hy either nilclowopy or cultirrc.

Ihc 35 control \ttx~l san~ples included IS from healthv 'trvinr ncgatlvc for conlmon rnteric pathogens. six I ' I I \ I I IV~ for cnteric hacteria hy bacterial culture. ten ~~l\ltlve for (;iorrliu inrrcrinalir ~ysk and four for egg\ 'I ~\~rirri.s lunthrirrtrdrr. All of these 35 st1r)l xamplcs uuru negative for f. hi.~rol,vricu. E dirpur, ;md I:' f~ro\h- 1 ' 8 1 $Air n~mplex cysts and 1n)phozoites hy k ~ t h mienn. ."pv and culture.

Iwsh unpre~rved stn)l samples wcrc collected in 'Icrllz capped containers for examination hy micros- [up) and culturc for Enramurba species Aliquots of !rr*h unpresewed stool samples were stored at -?(I"(' until used for K'K and ELISA tests

Illah saline and iodinc wet mountsof fresh stool sampler &Crc ~xamined mlerwc)pically for L /l~rIo/vlic~. f..

dispar, or fi mo,shkovskii cysh and trophon)itcs as prc- viously d e ~ r i h d . "

Stool samples were cultured for Enranioeba species in Lockc-egg medium ( N M modification of Rocck and Drtwhlav's medium) within 6 h of collection as previously dcsmibed."

Ncsietl PCH-resrrioion fiugmenr length polyntorphisn~

Extraetion of Enhmwba pnomic DNA. DNA was isolated from stovl spccimcns by the cetyltrimethylam- monium hromide (CI'AB) extraction method, modified from a previously described method for DNA isolation from in vitro cullures of Enramoeha.' The extracted DNA was passed through DNA clean-up spin columns (Hangalore Genei, Bangalore, India) to minimize PCK lnhihrtors and improve the amplification performance. 'lhe DNA was stored at -20°C.

Qumfiblion d DNA in slool spedmem DNA In the spin column purified DNA extract from stool spc i - men was quantified by UV absorbance using a Cintra 5 double h a m spctrophotometer (Bristol. UK). DNA yielh were calculated as IIV absorbance x dilution. l h e purlty of the nucleic acid in the samples was estimated hy the ratio of readings at 260 and 2Mnm (0DMJODN,).

Plimers n . R a d on the sequences of the 16s-like r KNA gene of E. hirrolyrica and E dispur, nested set 01 prlmcn (designated E-llE-2. Eh-IIEh-2, and Ed- 1IEd-2) wcre uxd, as described in 1998 by Haque t.1 al.." for detecting f,. hrsrolyrrcu and E. dispar in stm~l spcclmens In addition. havd on the sequence of the IhS-like r KNA gene of E nlc~shkov.skri Larcdo. a ncsted r t of primers (designated Em-11Em-2 and nEm- IinEm-2) was uxd , as derrihed in 2003 hy Ali et al..'" for dctcctins I.1 n~orhkovskii m stool specimens The primer sequences u x d for nested F'CR are shown in Tahlc I.

Standard s h i n s used. 'lhe three standard stratns E. hi.rnbricn HM-I: IMSS: E. divpar SAW7M: and E, nros11kov.vkii Larcdo were used as positive controls in this study. 'Ihe lyophilized DNA of these strains was generously provided hy Dr. (: Graham Clark of the London School of Hygiene and Tropical Med~cme.

Nested PCK-restriction fragment length polymorphism for E hisrolyrica and E dispor For a reaction volume of 25p1,comprhing 2 .5~1 of 10 x W K huffer (Bangalore Cienei). 1 .5~1 of ZSmMMgCl, (Banplore (ienei). 1 . 4 ~ 1 of deoxynuclemide triphosphate mix (5mM of each d N P . ABpens. Epsom. l J K )

K. Khairnar el al.: Nested PCH.HFLP for Lnranrocba

F* 1 Enrmo~Lm ntushkovskir-specilic nesled I'CH-RFLP. I IJd and even nunihered lanes represent und~gestcd and Xho- I J~restcd IT'H nnrluctr resa.ctivclv. 1.unr.c ID-.W. IINA . . lrom nod s p e c ~ k n showinp fi:. nn~.~/~kov~k,r: lonr ~ . ' a 100- hp IINA ladder

Yhri-l for I h at 37Y' according 10 the manufacturer's ~nrtructions (Promega). KFLY-digested product was u\ualizcd hy loading 10p1 of KFLl' digert on a 1.8% aparow g l containing cth~dium hromide. Xho-l exclu. h~vcly cut? (he 258-bp nested K K pruduct to p n d w e !6hp and 22-hp fragments (The 22-hp pnduct is not bs~hle o n the gel hecaure it was t(w~ small to he reullved ,111 P 1.8% agBf(Ke eel) (fig. 2).

I'nmer valtdatmn Ille wqucnccs of the primers E-I/E-2. Eh-I/Eh-2. U- I td-2, Em-IIEm-2 and ntim-IlnEm-2 to he urcd for ~dcnttlication of t, hnrolyrira. E, drsprrr, and 1;. nlosh- ii!vskii were lirsl subjected to a HLAS'I' search of the i'cnome datahase of all organisms (http:llwww.ncbi. nlrn.nih.gov/hlasll) and were found to he specific for the \ludy. l h e amplilied R 'R products of E. hrsfolyficu,

dispar, and E. nrushkovrkii yxcics in s t (r~l samples rcrc conlirmed by sequencing both svdnds of DNA w ~ l h an At41 377 sequencer (Indian Institute of Scienee. 1hnplore. India). All Jequcnm were analyzed for I*lmology hy using the nucleotide-nuckotide BLAST rarch feature (http:l/www.nchi.nlm.nih.g~~v~IastiHlat. (!I),

Ihe kknt~ties between the xqwncing nsulls 01 the IY'H pmduch of E, dispar, E. hisrolvrica. and E. ntosh- kwrhr with the sequences deposited in CirnBank ';l~cenion numbers 249256. h56991. and AF 14YU06.

respectively) were analyzed by using the HLASl sequence alignment (bl2seq) feature (htlp:llwww.nchi. nlm.nih.govhlast/bL2xq/wblast2.cgi).

7echLoh E. histolytica I1 ELlSA

' h e TechLab E hislolyrrco II test, designed to dctcct specifically the E. hisrolyrica lectin antigen, was performed on 45 stool specimens randomly selected from 202 patients with stool specimens positive for E hlrro- lyficu. E dispar, or E. moshkovskii by microscopy or culture. The '1'echLah E hisrolyrica 11 test was performed on stool specimens as per the manufacturer's instructions.

Anfiamorbic anfihody drrecuon by rapid indirect hm~i~~~luf ina r ion rrvr

H l t d samplcb were available from only 85 of 202 patients with stool specimens positive for E. hirrolyricu. E. dispur. or E. moshkovsk~i by microscopy or culture. Blond ywcimens were also collected from all 35 control group individuah included in the nudy. Venous blood (Sml) was collected in a sterile container,and the serum was separated and slnred at -2Q"C until used. A rapid indirect hemagglutination (IHA) test was performed on serum specimens of all 8.5 patienls (pnsitive for E. his- rolvticu. E dispar, or E nroshkovrkii in stool specimens by microswpy or culture) and of the 35 control group individuals included in the study with the method dcvrihed earlier." A titer of 21: 128 was considered positive k ~ r amoehiasisl*

Srurisricul dara analysis

Sensitivity wascalculated a\ the number of patients with positive tcst resultdtotal number of patients x 100. Specificity wascalculated as the number of controls with negativc tcst resulLdlotal numher of controls x 100.The agreement bctween the tests wascalculated using kappa stat is ti^^ 'The kappa statistic was obtained with Micro- soft Excel 2Ml.1. To determine the statistical significance of differences hetween the proportions, rsquared and Fisher's exact tests were used. 'lhe X-squared test was performed and the odds ratio determined with Epi Info Version 6 software.

Microscopy and c111rure of sfool

Of the IRO stool samples wreened. 202 were positive for k:. hisfolyuca, E. dispor,or E. moshkovsku by microscopy or culture ('bible 2). The mak to female ratio among these 202 patients was 1:I. All 35 negative

k. Khnirnar el 81.: Nerted PCR-HFLP for I:ntwrorba 6.15

Tnblr 2. Conlparison of results of nested K'R-restriction fragment kngth polymorphism (HFLP) and microwopy and culture ,,I .;~(wl spsomcnx /

Micn~ropy and culture result' - -- PDaitivc hy R~silive by Nogatlve hy microscopy mlcroxopy Positive by microropy Total no. of

w lcd PCR-HFLP result and culture only culture only and culture specimens . -.

f ilupnr (m~noinfcution) 77 IS 4 0 Y6 I Irr~rolyriee (mono~nfectwn) I I I1 0 0 I I ! nrorhkovskii (monoinfection) 2 0 0 0 2 I ihrpnr + 8 mr~shkovskii (mired) IS 0 1 0 I h ! (lopar t E, hurr~lwico (mixed) -34 I I 0 36 E h~~trdytrrn t f;. rnmhkovrkri (mixed) 2 n 0 0 2 ! ,Idspar + E hkro1ytir.u + E moshkovskii (mixed) 12 0 0 0 12 \ic~tive I I 6 111 35 62

l,!1,11 1M 22 I6 35 237

\I~o~wopy and cullurc alvkl no1 dasunguish among q%Ls and lr~1ph01011~101 A II~.~IO/~I~(~~. E dt,pal~.r. or E moshkovskir in s tm l EPC~MS

. ,~i i tr(~l s tcr~ l v~mples were negative k)r F;n~umoeho m z i o hy micrcwopv and culture.

i h ~ results 111 thc quanttlicatbn td D N A in the st(n)l $;krlmcns sh~rwcd the D N A yicld to he approx~matcly :.ip)llml. l h e purlty o f Ihc D N A extract from sltx)l .Filmens was sati\lanc~ry. as the Ol)n/Ol)L, ratlo was :oproximatcly 1.X Ihc xqucncing of the Pt'K p rduc ts ' I rlt.spttr. I: horr~lyttl-a, and I{, n t ~ ~ d k o v r k t t from rll specimens sh<~wcd reawnable identity w ~ t h thc

,tnl{ank srqucncch 01 ar~cssion nun~her ZJY2.50. . ~ r s ~ o n numher XShWI , and acccs~on n u m k r \ I I4UWd. respect~vely

Ihv nc$ted IY 'K-KFLI' was p r fo rmcd on a total of :' \ t (~11 spccimun* Including 202 stcxrl spcclmuns pal. .L lcnr t.. Iti.\wlvnca. I: (Itspar, or E. tfroshkocskri hy iirttwopy nr culturc and 35 nc~at ivc cotitrul stcr>l kt lmcns Ihe nested IJ('K-KFLP was ps i l i ve in 175 : ~ h c 202 \ t c r~ I specimens positive tor E hr.$mlvricc~. ali\pur. or I: nr~~.~/~Aovskrt tn)phon~ites or ~ys l s hy

lltrc)w~,py or rulturc (lbhle 2). PI^ a wnsitivity of 'It'b. N a t e d Pt'K.KFLP results fur rcpre~fnlal ivc [lid spcirnens are shown i n figs I and 2. ,411 35 ncgativc n lnt ro l stool samples that were ncga-

I\L hy h o h rnarr,u.opy and culture for t.'. ~~~~~~~~~~n. ' Bl~par, or E. ntorhkol~~kr l trophnubitcs or cysts wcrc ~iul negative hy nested K'H (TaMe 2). fur a specificity I llXl'?i,. Ihc prc~hahility of negative nested KR results i n the

" ncgstive control r tcn l samples being caused by K'K nhlhlturs was ruled out by spik i ig the D N A of the C~a t~ve st tn l spccimchu with standard Enfmtuchu "\A. lo lbwed hy n e a t ~ d K:R amplification.

'Ihc ncstcd PCK-KFLP detected monoinfection with E hisrolyriro i n 5.4% (1 11202 stool samples). E. drspar in 47.5% ( W 0 2 ) . and E. ntoshkovskii i n 1.0% ( 2 n M ) of sttr>l sample*'lhe ncstcd PCR-RFL,P detected mixed infections of t;. dispar with E moshkovskii i n 7.9% (161202). t.. dispur with E hrsrolyriro i n 17.8% (36R02). E. hi.1101yura with E. n~mhkr>b.\kir i n 1.0% (21202). and with all the three species in 5.9% (12R02) of stool samples ('lable 2).

I*.o stool samples were posttive for E. nioshkovskii exclusively hy nested PCK-RFLR .h is finding may explain thc prevlous finding of some of microropically postttvc hut anttgenically and PL'R negative results for E, hraol~rrl-a and E. dt.spar.\ugesting that E. mosh- kovskrr may he present in $uch strn>l samples

Ihe prvvalcnct. ol E wtr~shkovskri. E. divpur. and E. hrstol~rrre among patient5 at JII'MER hospital clinically ruspected to have gastrointestinal infections is shown i n h h l c 1.

l ' i r h l . ~ h I?. histolytiw I1 EI.ISA

'lhe li.chL;~h E. hisrolyrrcu 11 EL lSA was positive for E. hi.~rolynrrt-spec~fic lectin antigen i n 29 of 45 stool specimens posit~ve for E. hisrolvacu, lil d i~pur . or t,'. moshkovskii hy culture (Table 4). Nested K'R identi- lied E. hisrolyricu D N A i n 32 of these 45 culture-positive stool spc~imens

Compar~wn o f results o f the TechLab E h~srolvrica 11 EL lSA and the w a r d K:R-RFLP on stool specimens showed that o f the 29 specimens positive for E. hufolyrica by the antigen detection ast. nested F T K deteaed E. hurolytica DNA i n 27 specimens (17 p i - tive for hoth b:. hufolvrrra and E di.vpac o n p t t t v e br hoth E, hirrol~rrca and E moshkovskii: six pcaitive

hih K. Khairnar et al.: Nerted PCK.KFLP for tnramorbn

.ruble 3. Prevalence o l Enramurba dispar. E. hisrolyrica. and E. moslrkovskii among prlicntu presenting at the hwaharlal lnstitutc o i Postgraduate Medicxl Education and ~<e\earch (JIPMER) hospital clinically suspected to have gastrointe.;tinal infections

Iype of No. of samples positive hy Prevalence inr,munaba .yxcler nested PC'R-KFLP . -- -

t nrushtovrkii 32 1.9 t drrpar 1HI 9.3 t hurolyriro 61 3.5

lo~rl no. ol a w l rnrnplcs tneludcd In the Budy = 1720

Tvbk & Compariw~n (11 rcsullb of culture and the kchlah I< Ir~.sn,lyrico I1 cnzyme- i~Lcd immunow>rhcnl may (ELISA) performed tm stool \pecimcns

rcchLah L hissi1yric.o I1 \,# ol patient specimens with st(xrl spclmcn result' r~rction ~n culture krr T h l no. of i ,iremurho It~\i l ive Neealive \mcimen*

t hnndvrrca II El 1SA 1% k s y r d lo &led only t horolvtico-rpccnfir Irclnn ;tn~mgcn ~n <tool ~xolmrn and rhcrrk~rc u n a,drm only the prcrrm ol f. hrrrolrrn.n t htrrrdvr~~r was Jnmlrd hy PC'R on 5 of thcx *.lo antlpcn ncpalivc 51ool .ipcclmens i h,~rolvrtro war &,lrrtcd h) H'R an 27 of lhuw ?V anllgn ~ n n t l v v ,1001 yxcuntn\

I rbk I ('umpar~u~n of retultr rlf nested I'C'H-KFLP andkchL3h L. h~crolvrica II ELISA un stool specimens

TechLah E hbrolyrrca II stiwl rptximm test result

- - .- . 'lbtal no. of \<,led IY'K RF1.P ru\ult

.. .. .. - - - . . . . . R,r~tivc Negative \prcimen . ... .- . - . - - --

I rlrspor i m o l ~ ~ ~ m l c n r ~ n I 0 7 7 hinolvrtcu ( r~~on~~~n lcu tn~n) 3 I 4 nr~rhkurrk,, (mont,inlecuon) il 0 0 ibrltur + L nr,~rhAr,vsAa Imlxvd) il 7 2

i ,llrpar t I, l~os,lurtt~t (mixed) 17 I 18 i h,~roIyrr~a + f ' mo~hk,~\,,vkii (mtxed) 1 D I 1 dtcpar t E havrlrrrtrr t t. nrr,~hkra.,lri Imlxcd) h 7 Y \cgsllvc 2 7 4

I 81ill 29 I6 45

I l ~ r C. hrrrolyncu. I:. ~r~r~Jtkov.cka and I< disprrr. and !hrre positive for I:', hrsc~~lynra), kw a c t ~ r r e b l r ~ n of 'I' I (Table 5 ) .

Ihc rapid IHA test was p d t i v e lor antiamoebic antihdy i n serum specimens of only 19 of &l5 (22.4%) Ntlcnb pusitive lor E hbrolvnca. E dispor,or 6. mosh-

I ( ~ ~ u k r i in stool specimens by microscopy or culture. O f Ihcu: 19 rrropri i t ive patients. 12 (63.2%) had slool \itcimens p c ~ i t ~ v e for I.:. hrsrulyriru (as mono- or mixed

infections) by nested PC'R-RFLP. wherear 31 (47%) of MI seronegative patients had stool specimens positive for E hirrr~lyrica (as m o n o and mixed infc~tions) ('lable 6). One (2.8%) of 35 control xubjece was prsitivc for antiamocbic antibody by IHA o f serum.

Comparison o f the results of stool kct in antigen detection with serum antiamcxhic antibody detection i n 4.5 patients showed that of the I 1 seropositive patient& six (54.5%) had stool specimens pusitive for E hivto- Ivcica by the antigen delection test. whereas 23 (67.6%) o f 34 seronegative pattents had stool specimens positive for E. hirfo!vtica infection ( 'hb le 7).

h Khnirnar et al.: Ncrted R'R-RFLP for fi:nrurn~~bo 637

nMc 6. (:omparison of the ncaed PCR-RFLP resulls prformed on stool specimrns and anliamoehic antihody lest result in ,rium nf patients presenting al JIPMEH hospital with pastrointotinal di%umfurt

No. of specimens with rcactton for antiamocbic antibody in

seruma .- T(>,uI no. of \~,tcd PCR-RFLP result Pusitive Negative specimens - .- -- I dnyar (monoinlcc~icfion) h 25 31 i linr~~!vrkrr (muno~nfect~on) 4 3 7 t , n~r~hbov~h i i (munuinfection) 0 2 2 I ,iiy'or + I: n11whkev.skii (mixed) I 3 4 I rlrtpor + i. hiralyrau (mixed) h 19 25 1 l~~rroly~ico t L morhkovskii (mixed) 0 1 1 ! ,ir~par + E hirn,lyriro + E mrhkovrkri (mired) 2 X 10 \LC.,IIV~ 0 5 5

18111 19 66 US

rlum ~ndtrcct hcrna~luiin81,on tttcr 2 I :I28 wus consulcrcd pn~i~vc

IrNr 7. Cr~mpnriu~n ol the 'rcchLah fi. hirr~~lvr~ro II ELlSA rcsulls prbrrncd (rn ,w1 y rc i mns and the antfiamcvhic rnrihody test result in serum of pataents present- i dl JIPMkK horpttal w~ lh pa~lro~nle%t~nal discomfort

~kchLah E hrsrolyrrco 11 \ CII pxinicnr with sl<wl spec~men lest rcsult ;~.r>un lor ilnl~dni~wbu' .. . . 'Ibtal no. o l hdy !n *rum Ih\tlirc Negative specimens ... .. . . - . ... . -. - . . . - . . , .. .. . -- -- -- . .. -

t'*,l,w h 5' I I \~E.IIIV~ 23 I I' .14

"1.41 29 I6 45

I"*.* IWCS \I'r,t \ p t ~ w n \ po\tt~rc fcsr f hnrohato hy maeJ Y('R.RFLP IsC, lhwc ucwl ycrtmcns pnnln~ for I: A~rrolmru hy m a d PC'KRFLP

he ahlc tcr dctcct und disttngu~sh 1:. h~.rro(vricu, t. ,~'irr. and E. ntoshknr~kri in stcr)l ranlplcb ir extremely

"'pc,rtanl for accurate d ~ i ~ g n ( n ~ s u f lntc\tinal amoehra- .dnd for knowlnp thc true prevslcncc of pathogenic I ~ r ~ r o l ~ r r ~ o lo a community.' I n recent vcan PC'K

:i. heen ~ncrcarnglv uscd for routine diagnosis of - m y infectious drsenscs including parasttic dirases b1 cxploitinp; the y n e t r differences hetween 6 hblo-

and t.. Jfspur,reveral rcwarch groups have devel- :id K'K-hawd a w y r for the dwrtm~nation of these i'll !pecie~"':' Most of these R 'R analyws are h w d I llle use of scqucnces o l the extrachrtmosornal citcu-

4' rKNA gene. present in ahout 2lKI m p i t ~ i n each 'olnmrr& .buck'.'::' " .\ few rcpcrm have conuJered E. n~osl~kovsku along

'IIh I: hurolyr~co and E. dirpor lor detection in stool Wclmens hy WU.:."" In the pmscnt study, nested

k was negative in 27 of 2(!2 stml specimens poRiIive llq ~:n/umurba species tnyhozoilus or cysts by micros- % or culture (I'ahle 2). Ihe negative PCH result in k~ 27 st1101 samples may k due to the presence of

other Lnran~oehu species or may he imputed to a limila- tion of the sens~tivity of the nested Pt'K technique.lhe nested R'K-UFLP results reported here showed a sen- sitlvrty of 86.6% and a specilicity of 1W%, indicating that II IS a dependable methrnl for specific detection of h:, IIISIOI~~ICR in stool specimens

'Ihc werall correlation hctwecn the nested PCR- KFLP resulu for stool specimens and those of antigen detection tests for detecting E hisfolvrico infection was greater than YO%. Tbis agreement k tween techniques providesconfidence that any one of the techniques may he 11.4 alone to accurately assess the presence of E. hrsfolvricu in a stool specimen. Nested PCR-RFLP has the avantage of identilying and differentiattng all threc morphologically similar forms of amoeha. E hbfolyrica. E. dispur. and E. nroshkovskir, w h i h is not possible with ELISA or culture. Our resulu clearly indicate the superiority of K K over ELlSA for detecting and characterizing the species of amoeha at the D N A level.

'The prcentage agrcernrnt between ELlSA for E. huru(vaca lectin antigen detection in stcml specimens and I H A seropsitivity for antiamoehic antihaly was

K. Khairnar el al.: Nested PC'H-HFLP k>r I.:nlrr~wocha

!7.78%.'lhe kappa statistic was 4.W, which indicates pmr agreement hetween the two tests

We also found no significant association between ~cri)p~silivity for amcwhic antihodies and stool ptrsitiv- ~ i y for E. hisrc~lyrica by ELISA. In thc present study. 20.7% of the patients that were infected with E, hisro- lvrrcu wcre seropositive. and 31.2% of the patients *hose stools were L hisrolyrica-negative were scroposi- itve (Fisher's exact test. P = 0.483) (?hhle 7). Ertramoeba I~nrolv~icrr lcctin antigen was detected in s t~ud spc i - nlenr from 23 of -34 scronegalive patienls ('Fdble 7). We rlro found no significant as%riation hctwecn wropusi- ~lvity and pcnitivity for stool infection with t:. h&rolyrirn h! PC'K-KF1.H In the present study. 27.9% of the patienls inkcted with E. hisrolyrica were seropositive, ,ind 16.7% of paticnh whose stools were t:. hi.srolyrica- ncgative were seropnsitive ( x 2 = 0.27. P = 0.603) (Table 11).

lhesc results contrast with thosc frwn Hangladesh. uhere it was found that 52'% of children colontzcd with 1 hirrc~lyrica were seropcflitive.' Our data alw differ Inrm th tw of a study conductcd in India that found 12.8% of wnrpnitive individuals to he colonired with .In E hirro1sric~1-t.'. dispar complex (as detined hy mrcr~xopic examination of stool). whereas 20.3% of mrnegativc individuals wcre colonized." Kcsula milar to o u n have heen reported from I3rlvil. where nil ct~rrclation was o k r v e d ktween seropcdtivity and \IWI colonirati~n with E dbpur or t:. hisr~ljrica. In the Ilruilian study. 19.5% of the individuals colonized with 1 hr.srolrrn-u wcrc scropcnitive:" this result is similar to ( u i tindings In India.

I ' rev~~us reporb hugest that diffcrcntvation by ~ulture/~.urnfymc analysis or antigen de!ectro~r kits might ~,vcrlcu~k mixed infecttons with E, hisrolyrtca and I rlrsplir. since such ~nfections were dclcctcd only after I'I'K war u\ed directly on the u m c stcml spcimcnh i'rcviously ill.sayed.'!." Kegardlnp antigen-detection ':r~th(KL\ a small n u m k r of I.. dispor organisms present ,$nlr)ng a large ppulation oft.: hisrolyrica would he dif- rltult to detect using an t:. hhrsrolyr~ccr-specIRc monm~lo- fir1 antilxxlv or a monoclonal ant ihdy cross-renctivs vih E hi.rrolyricu and E. :.;spar In such cases only Ihc usc of IY'K or an E, h~srolvricu- and 6:. dispar- 'pccific monwlonal an l ihdy would cnshk diffcrcntia. Ilon. ' l h e r ohwrvations may explain why mtxed ll~lcctions with t; hi.srolyricn and t.', clupar were not iil~rrted prior to the dcvclopmcnt of morc spccilic and \in\ttivt: molecular techniques

In the present study, wcult infection (I'C'K-pcrsilive hut ELISA-negative st(x11 specimens for L hisrolyrica) QL\ ~ k r v e d . Occult infection may p~ssihly he caused hv degradation of lectin antigen in stiu~l specimens after Pnrlonged storage at -20°C' hefore testing hy ELISA. whrh might result in EL.ISA negativity in ITK-positive

stool specimens The percentage agreement between ELISA for E hisrolyrica antigen detenion and Pf'R for t,'. hurolyrica DNA detection in stool spcimcns was 82.22%. 'Ihe kappa statistic: was 0.60, which indicates moderate agreement hetween the two tests

This study included patienu from various age groups and from different geographical localities which shows the wide distribution of E. moshkovsku and E. dispar in Rducherry and its environs ?he study has several interesting findings

First,of two patients with E moshkovskii monoinfection, one suffered from dysentery, although the associated bacterial etiology hy routine stool culture was negative. Other investigations. such as viral studies. could not be done in our laboratory. ' h e high prevalence of E. moshkovskii among the study population supporn the view that humans are a true host for this free-living amoeba and arc not just transiently infe~ted. '~

Second, this study highlights the risk of relying on microscopy for the diagnosis of amoehiais Of all patients with gastrointestinal discomfort diagnosed with amtshiasis by microscopy or culture, only 30.2% were proven to have E. hhrolyrica infection when nested PC'K-KFLI'was used.The study showed an appreciably hifih prevalence of E. (fupur in the patient!, compared with E hisrolyrica, indicating that only 61 01202 stml samples with organisms resembling E. hisrolyrica hy microscopy actually contained E hisrolyricu, implying that the majority of suspected infections are midiag- aosed and might be treatcd unnecessarily with anti- arnwbic drugs when diasnoscd on the hasis of micnwopic lindings alonc. Other invcstigators have also found that infection with E diqpuris more common than infection with E. ~~isrolyrica."~' One report. however. claims that infection with E. hi.srolyricu is pre- dominant in a population."'

Ihlrd. the prewnt study showed that the rate of monotnft.ction with E. ddisyar was highest (47.5%:961202 \tml samples) among patients presenting at JII'MEK hospital. The study also shows that the rate of coinfection of E hisrolyrica with E. dispar is highest (17.8%: 3N202 stool samples) compared with E. dispar with f.'. n~orhkor,~kii (7.9%; 16/20? stool samples) or E. hisrolyrica with E nioshkovskii (1.0%; 2RC2 stool samples). Coinfection with both E. dispur and E. histo. hrica in the same stool specimen has heen reported ear~ier,12.1* l*J1.*.dI41

In conclusion, epidemiologic studies and clinical dtagnosis of E hisrolytica-associated infection hased on morphologiial examination alone are prone to error. Infections due to both E dkpar and E moshkovskii are assuriated with an asymptomatic carrier stage. P('R i s therefore.essential for distinguishing E. hismlvricrr from E. dupar and E. moshkovrkii.

g Khairnar e l al.: Nested K 'R-RFLY Rlr ~:nrnntocba

.~ , I rmwl rdgmma Wc thank DSTE (Department of Science. ~~chnology and Environment), h d u c h r r r y and I C M R ~nd lan Council o f Medical Research). New Delhi.fnrfunding ,hs pmjcct. We sincerely thank TechLah. Ine. (Blxkshur& \';, ) for generowly providing the TechLah E hbn~lyr ica II 11 ISA ter l k i t free of crat for the p u r p of research. We ,IN, thank Dr . K.V. Kirubvankar and Dr. Gowrisankar from i)~parIment o f Preventive and Social Medicine, Jawaharlal ~~,?l l lute of Postgraduate Medical Educatinn and Research. i'uducherry, for doing the statistical data analysis of this *,,rk.

World Hcnllh Organomlion. A m u b m ~ r WHO Wkly Eplemnd KCC IYV1:RV.I. I M ('lark ('(i. l>amnnd I S T k hcr& stra~n and othcr Emomwho b,~lolvti~o-l~kc r m w k arc f;nrann,rhu moshkomrk,~. Ma1 Ilarhcm Ih~r in~tnl IWI:4h:114. l ~ ~ ~ n r a l c ~ - K u v A. H y u c R. Ayu!rrc A. ('astanon (;. Hall A. Iruhl F. c l 11. Valuc of murovnpy ~n the d~agntnn of dyxntcry .rwr~ulrd wnh Invnnve tnromrxho hurro1v11t.a J ('tin Palhol l'FU,47:2)h V I1art1a K'. Amwhac lntcstnal amcchx nath<wnr frcc-ltv~nc . .. .~rnochac In I 'ar~lr St', rdacx. Tcxl h w k of medacal paras~b>l<yy i~wul~rrtu~lnpy and klmsnlhdqy). ('hcnn~~n All lndm PuMnhcn a I>t\lr~hutwr. lm p. 2'4 h l . Lrtwlad I ) J . S ~ l n r r H(' 11. Hcaly (iR.(,kru>n NN.katon DJ. Ilern>t> ('A Amwhaur rpldem~obpr \tud#r\ ~n thc l ln~ led tlrlrr. 1971 IU74. Ann lnlrrn Mcd lq78:M 8'1- W Haqw W. Ncvdk Lhl. tiahn P. Pctn WA Jr Wapld dlapn<nar of t nmamrho ~nkovan hv u ~ n p tnrom,rl>r and tnrumochu hrmt,. !,lrco rl<wl anllgcn Jclcd%on knlr J ('lln hirvohol IWT.1l. :($x n l . llu4tw1 (I). Haquc R. Prlrl WA I r kl~, l r rulrr-hard dugncntr '81 f~sum<rho h,srdrr~ra ~nlrcuun trpl.11 R o \lo1 Mcd "NV.2.' I I I llrquc W Ll<dlvh 411, All IK. Alam K. L.uh;~nkr .A. l.ycrl) D.cl r l I )~agncr~r 111 awtw llvcr a k r s and ~nle\ltnd ~nlectfun w~th lhc Icchlah f nrumwrha hurolvnc.~ II anltpn C l e o ~ o n and ante. IrJy ~ c u \ I ('lm Mcrdnol Z(UI.UI 215 V lurnw\ U. M w J ) AH. ('ho'd~nn I'l. ('amptt\un 01 K 'R and .%nt!grn h l r f l r t n mclhvd< lor ddlapn<n8sol I nlomcul~o ltrrrt,l,rw nlrrt~c,n I ('Inn Palhc,l .YXU.5I.I?hl h l,,ann I: Irmk'l I. I k c c i ~ o n and dnflcrtnlt.tlnc~n ol t n a m < w h lii*l~,hran and fnran~,rrhn d u p r nolalcr In c l ~ n ~ n l rrmplc% hv IT'R a d cnqmc lnnkrd ~mmunnwrknt a s h ! . I (' l~n Mlcrohul '1111.41 217 41 \harms AK. (hahhr .\. Hansal (i. K ~ u r 11. Vohru 11 Evaluatbon I nrvcv d t ryan l r tnclh~xk fur thc delrcllun a d d~ l f r rcn t~u t~~m ' I f.ntomrut*r hunrlvncn In an rndrmw arc. rrm\ R Suc 'Rcy \Ird H)? I(KItV7 1%-7 llnquc R. Al l IKM, Akthcr S. PEI~I WA JI ('ompartron of IY'K. kurn ,ur ianslynx m d rnllgcn dclrtr!m fur dlagorls of I ~lum,xlur hr~rc~lvlrru mfrctr,n J ('lm Mcr~,twd IWH.WUF

l'.#rqs X'. Kha~rnvr K Lnwmnrlu~ ~ ~ ~ ~ A o I I ~ I I and t n l m w h . l tb r -swn.ud lnfccI8onr in H>drln.rrv. Itnltu. J Hcdlh Rlpul hult X!iJS.U.M 5 'rrlla S('. Ynhhnkrr PK. Evslualton 01 Irct<b-phcnol collon hluc 1111 w.1 n ~ o v n ~ prqwatan of kczs J ('ltn M ~ r o b c ~ l IW5.11: ~llI~J.71 Yarqr X'. Rao RS Stat1 rullurc 8% a d ~ a g m l ~ c r ~ d In thc delcc. lltln of I..ntm& h~~dtnlv~ir. in ~ h c faecal ylrrtmcni Indian J 1'4thel Mrrcrhol IW(:.M.35%63

Ih. Ali IK. Hasain MB. Roy $ Ayeh-Kumi PF, Pctr~ WA Jr. Haqw R, el al. Entnmorba mhkovski i rnkctlona in children. Hangla. dcsh. E m r e lnfcci hr ZfN?3kW.

17 R r ~ j a S( ~a,lnathan I Rao RS Rspd tnchrcn hsemapelut~na tlon frapld IHA) ustnp renrotncd chrk cclh for urudiagnosn n l amuti&ir at health centre kvcl. J Trop Med Hyg 198992:221-6.

18. Parija SC. Kasinathan $ Rao RS Comparalivr evaluation of mlvxcnic and a x n e amoebic ontieens in indirect haemaanlutina- ;ion (IHA) for xrodiagmn~s of rmocbiuir Indian J M C ~ M ~ O -

hiul 1988,6:87-97. IV. Acuna-Soto R. Samuelwn J. De (iirohmi P. Zarate L. M~llan.

Velssco F. Schoolnuk (i. el al. Application of the p o l y m n x cham reaction to the cpidcmtolqy of p thqen ic and nanpalho- p n r Entamorbo h*rdytIra. Am J Trop Med H y l 1993:a 8 7 0 .

20. Agulnc A. Warhursl DC'. Ciuhl F. Frame IA. Polyrneraw cham remion wlulion hyhndualmn enzyme-linked immunonray (PCRSHELA) krr the diffcrmt~sl diagnosis of pathogenic and nonpathogenic tnromoeba kt~~ololyrico. Trans R Sof Trop Med Hyy 19P~R1181-X.

21. Clark CCi. Diamrlnd LS Rihc~omal RNA genes of "pathopnr' and "nonpathogenic" Enramwho hisrol~rico are dirt~nct. Mol Hiochem Parasitol 1W1 J9:297-112

?? Kal,wonlrl W1.ddrr.h I k h e r r R m k r II I>,rrfl amplob tlun dnd Jnflcrcnt~atn~n o l path*m~r and n~nnplhogmc Fnr marnu nrnnhnra DhA from ,t<wl swzamcn\ Am J Inx? Mrd Hyg IW451:115-8.

23 Novat, S, Stront M. Granata S. Bruno A. (istn S. Scadla M. el al Dtrcct xquenctny of !he W R ampltfied SSLl rRNA gene of Enwnwh~ha dlrpvr and the drug of pnmers for rapid differenoa- tlon from Emmotha hmolvoro. Parar~tolvgy iW.1 1?:36>9.

24. Tachnhana ti. lhnra S, Kohayilshl S Kaneda Y. 'lhkeuchl T. Walanak Y D~ffcrrnccs ~n pnomtc DNA xqwnc.-.i hlween pathlgcnr and nonpathogenac ~wlales of Enlomoeha h a r ~ ~ l ~ o a ~dcnttfietl by lalymraw cham remlon. J Clln Mlcrobol IYJI. m22.u Y.

25, Tann~h E. Burchard GD. D~ffcrcnttatton of nathomnlc from , . nunpathogenic t.nlam& hr~rolrrrcu by rrslrKllon irapmcnl anal~rs of a ,~nylc gene amyllficd ln nso. J (7x11 Mcruh~ul 1991. n.nn 5

26. Bhaltachavy S Hhallarharya A. Dsamond 15 ('nmpnwn of ~rpcalcd DNA bom ura~ns of E n m w h a hrr~olnrm and o l k r tnlmorlra. Mol Btnchcm Parsutol lW,27.2574?

27 Bhaltacharya S Hh;btlichaya A. hamond L S Snldv 41. Clrcu. lar DNA o i tnrmrwhu hua,hara cnctuler rnhommal WNh. J Ro107~ml 19RVRV)h:45L8.

?U H u k r M. Kollcr 14. (;tiler ('. Mlrclman D. Revel M. Ko/unhlau S et al. Enrumwb~l hrndsnt.n r~h,romal RNA wnrs .)re mrrad on pltndromtc oruular DNA n~oleculrz Mol D ic~hrm hras~ lo l IYXVJ?.3S-%.

20. f!amachu&lrsn S Hhatlachrr~r A. Hhattachsrva S Nuckotdc rgurnry m a i m df t k rRhA o a m m p t ~ ~ ~ unll of a pathuycna t n r m t w h hul,.I,L~o Urrbn I I M I IMSS Uu.'lcl' A.n+ Rcr Iw?2I :mI l .

10. Lhpal D. Mittal\: Ramrchandran S Dhar SK. Hhallachrr\a A. Rhattacharya S NwkotiJr xqwncc organlsallon and ~lmlyus 01 Ihc nucku nhru,mal VNA nr& ol thc proloruan praslte Enramwhu hirrolrrrco Mul Bluhcm Pnrulol IVUI-.67:?(LCI?

annam~xhr antibodrs rrlidinp in a hlpcnm!cmtc ,one. Scand 1 lnfcn D s IWIL1:771-6.

12. Brapa LL. Mrndonca Y. Pawn CA. Saks A. ('hvakantc AL. Mann ill. S c r x w u v ~ l v b r and ~nmnnal mlonizat~on w~th Enr. -rho hufol&co and Enram~xho dupor on ond~rtduab In norlh. rrslcrn H r u l J (18" M~crohud 199R-b .W-(

3.l RII.8 DR. K c n t o ~ 15. ShcpolnJ DC. Maclean JI) MrrPhcrum DW. Kuln KC. Lnromor(r, h&olrtrca awl Enramorhd dtpnr cpl-

dem~ology and compariwn of diagnostic mcthmb in a rrting of nunendem~cily. ('lln lnfcn 111s 199Y9V1.115-X.

U Rch~hna tl. Kohyarhi K,'Takcuch~ T, Fujiwara T. Prcpnralion ~ r f u m~~n~r lnns l anrlM) rp.c~fic for titwmabu dirpur and II~

ahlily lo di*ingui%h F dt~pur from E ht~tnlvrtco Clin Diagn Lnh lmmunol Im4:4W14.

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a (iathiran~ V. k k m m 'l+ Frcqucnuy dtstnhur~on of Entumorho h ~ v u l ~ c u ~ymtr*mcs Ina rural Soulh Afr~can p~pulaion. hncrr IVWS.I 119 21

K. Khairnar ct al.: Nested PCR-KFLY for Et~~anrorhu

19, Martinez-Garua Mf:, Munoz 0,Cjardwo.Ralriyu.r G. Sanchez- Pares ME, Valadel-Salazar A. PalaEiol-Beristain 0. ct al. Path(* gentc and non.pathogcn~c rymodcmes of Fnrumnrho h~~tolyricu In a rural arca of Mexico:concnrdanu: with xrolosylosyAah l n m l Med 199021:147-52.

40 Ciatti S, Mahdn R, Bruno A. Gvini C, kaglia M. A survey of amnhic infection m the Wonji area of Cxnml Elh~opia. Ann 'lioo Mcd Parasllol IWB92~~:17>9.

41. ~crulon.~nchcz OA. Sturm.Ramircz K. Romcro-Zamnra JL, Sanlos-Reoado JI. Samuelwn J. High rare of occult lnfcnlon w~th Enwmorbo hinolvncu among nondyscnleric Mexican chll- drcn. Arch Med Rcs IY979X:311-3.

42 Romero JL. Dzwteaux S. Reed S O r o w E, Ssnros J, Samuel. u,n J, (1% of oolvmeraw chain reanion and nonradiodflivc DNA prohs lo J L ~ W ~nmm~vbu h~~lol.vrico in ciiilcal ~mp les Arch Med Rcs lW93.277-9.

43 Oatt~ $ Cevin~ C. Rrunn A. Ramsan M. Marchi L. Scaglia M. Rnt irolatinn and charactrruat~on In humans of EnIuma.h? hts. tolrrrca (laboratory made) ~ymalemes XX. Parasitol Rcs 1W.83: 7 1 M

Research article

A novel nested multiplex polymerase chain reaction (PCR) assay for differential detection of Entamoeba hlstolvtica. E. moshkovskii and € . - tiispar DNA in stool samples Krishna Khairnar and Subhash C Parija*

U d r m Ikpdnrncnt of M~rmhiolw, lrwaharlal Innltuw of I'ostgraduaa Medical Wucnlaun and Rmanh (III'M1:R). Puduchm).. India

I rn.rl. Krirhotr Khnorn31. bikhr~rnar(ayaha, ro.in: Suhharh (: I'unla' - [email protected] 'I ~mmpondnn~ author

Abstnct B8ckgmbnd: E hmc+iKn. a pathogent m h , is indisongusW in hr cp t md vophozoite s r y s from h e d non-pathogenr E morhkorJv and E d4por by light microscopy. We have dndopd a nested muhpkx PCR targeting a IbS-like rRNA gene for dnlerentul detection of aH the three mcfphobguully s~mbhr f o r m d E hatc+wa. E morhkmkb and E dkpn simultaneously m stod umpb.

RerulCr The specles speck product smze la E hrndyflco, E inahkw and E dapor was 439.553 md 174 b!~ rewctwek. which was ckartr dillermt f a ail the thrn Enramocba rrrcler. The nuted muklpkx'PC~ sh& a sensatlvotr of 94% and spcificlty of 100% for d e k t r a u o n of E hmo@ca. E rnoshko* and E dspor DNA on stool umples The PCR was perm fw E hrno@ca E moshkcnkn and E d m m a total of 190 out of 202 s twl smclmens (94% senme) that were

cukure All the 35 negaove control stool samples that were negauw fw E h m a 1 E dlrpor1E m m h k a h b* mkroscoor and culture m e alx, found n m u n br rhe nested multrdu PCR I I W% " . speck) The result from the study show h t onty 34 6% of the p u n t st& vmpkr that were pcsitln b r E Mc+iKo/E daporlE rnashk& by eumlnaoon d stwl by murowopy and/or cuhure, were mw l t y p m e b r padqen r E h6rm)lrca and the renuinlng myorny of rtK s t d wmples were powtwe fw non-pathogtnr E dnpcn a E nwshk& as dcmomtnted by the use of nested mulupkx PCR

C o n c l u h The present study reports a m nested muhrpkx PCR s m t w fa rpecws rpeclhc detccwn and Merenliluon off h m w a E dapor and L moshkoda D N ~ m stool spec~menr The r a t n hghty speck. senuUve and also npd, prow&# the r a u l u wthm 12 houn of recemvmg

~ a c k g m d annually 1 1 1 . E. huw~ynco. the pa thwn i c amocbk is '~lfrction with Enwmnb. hwolyaa results i n 34 mil l ion indistinguishable i n its cyst and uophowiie stages from I I 5U million symptomatic cam of amorbiasis worldwide tho= of non-pathogcnir E. drrplr I I and E. mmhlmv*il "4th year, c a l u i q 40 t h w u n d u> lW thousand deaths 121, a c c p t i n rarc usts ofinvasive diseau when E. hurt+

lyrscu trophouri~e may conlain ingested red b lwd cells 111. Estimate of intestinal E. hisldylicrr infections have heen primarily baaed on microscopic examination of s~ools, whi& has a semitivity of only 60%. wen under optimal mndards 14.51.7hc microscopic examination of $id alone, however, fails to differentiate E. &Mu1

bom those of morphologically similar non-pathwic specie auch as E. dispuf and E. moshkuuskil. Stool culture iollowed by ismmyme analy~ir enables the diflerentia- tinn of E. h i s l o w fmm E. dispn 161. Howwer, this method of culture when followed by iaoenzyme analpis irquim one to wen1 weeb lo obtain the reult and also yerial labontoty facilities, malting it impractical for use In the rwtinc diagnosis of intestinal amoebiasis.

barious approaches an k i ng followed for specific idmti- tication and detection of E. hutolynca in stool specimens such u detection of E. hiclotpica roproantigcn in stool samples by enzyme.linked immuno-wrbcn~ asay (I.IJSA) and damion of E, hiswCtca DNA in stool wm- pler by polymerase chain reaction (PCR). Currently few .ommercial ELJSA kits are available for detection of E. his- :c~lmm/E. dl~par copmantiwn in stool. These include tihlrb Fnramnbn 1-1 l od i tm E hsrolpldlF d q r IS]. ~lzxon PmSrrrl FIISA to detect F hurolvr~mlt dsmr and . . . i mnlu hmblu 171 andTnage parasile panel to detm anti. ,rn of E hsroiynu/E dupar, Cmrdw Inrnbl~a and Crypt- ,~plwwlrum p n m m in stool rpecimens 181. 'The main I~mrtatson of all thoe EUSA kiLl is chat they can ihtirr ihr amoebae only as E hufdynu-E. dupnr compla but it>^ sprcifirrlly as E hisfdynu. E. dirprrr or E. mdtbovrldi. 'l~nvwcr. a monoclonal antibody buLdTech Lab E. hum flu I1 LIJSA IS commemalty available for the specific itmtion of I ' hutdpirn antigen d im ly in stml sped- rlrll 191. hut this kit can neither detm E, dirpnr nor E. .*<q*hkrnh~ tn stcwl sperimm.

Recently, a nested PCR targeting I6Slike rRNA gene has k e n reported from the Inumacional Cenve for Diar. rhoeal Disease Research, Dhaka. Bangladah (ICDDRB) 121 as well as from our labontoly at lrwahaltal Imtimte of Postgraduate Medical Education and Remearch (JIPMER) hospital. Puducheny, India 1101 to dctert and differentiate E. histolytica, E. divpar and E. mmWmvsCii directly in stool spedmens. But the identification of the amoeba in the stool specimens either aa E. h is row, E. dispm or E. mmhbovrbii was carried out by puforming nested PCR each time separately for individual species which was tedi- ous. To overcome this disadvantage, the main objective of the present study was lo develop and evaluate a nested multiplex PCR targeting the 16s- like rRNA gene for simultaneous detection and diflemtiation of E. hinolyt- icn. E. mmhkwskii and E. dtsprr directly in stool samples.

Results h 4 k r o ~ y ond whue of noel A total of 202 out of 1.720 stool samples screened were positive for E, hiswlytlca/E. dispar/E. rnmhLmnkii by microscopy and/or culture. These included 164 specimens positive for E. huro+/E. dispar/E. mahbanlrii by both microscopy and culture, 22 positiveby microscopyand 16 positive by culture (Table 1). All the 35 negative control slool samples were negative for E. histolyrh/E, disprr/E. mmhkmbi by microscopy and culture.

N"dIl)(l(llpkr=q ~ c f D N A m s o d r p c a r r n lhe DNA yield war found to be approximately 49 &ml. 'The purity of DNAavact from stool specimen was found to be satisfanor,' as the value of ratio of readings at 260 nm and 280 nm (OD ,,JOD was approximaiely 1 8.

l&f ~ r m p u k o n d r a * l d - ~ K I ( ~ C h . h ~ c t k n ) , m i c r a r o p v u l d c u ( N l . a I ( o o l r p c m m r

Muted PCR muk Mwrmcopy ad cdwn IcIUII. T d n r r d - -- - - --- - - -adbC~vllw=w MhfLll z,~s W - u d W ~ = ? ~

. - - ---. - .- -- - - - 'Ikpor(mOnOn(mm) m0 I b 4 0 IW 'hno)u(nom-) I1 1 1 0 t s I mihb%h(mnonkcum) 1 0 P 0 1 L I k p o r + L m m w n b ~ m i . r d ) I b 0 1 0 I8 i*+lhsohao~mb.d) IS I I 0 3 I ' h ~ * L m h k c d ( n h d ) I 0 0 0 1 L ~ . 9 m w w * - ( m b d l I S 0 0 0 I S %h 1 1 a 1s 4 1

- ---- luul 1b4 11 I& 31 111

- -- k r ~ ~ d c u h u n r u u u b * ~ ) d r t h ~ ~ h b r ~ c ) l s t n d ~ d E ~ L ~ n d L ~ m n m l ~ o . r m n ~

RmerWMotim Ihe sequencing mu11 of PCR product of E. dbpr, E. hbm. l y h and E. mmhkovrh'i Lircdo showed 19% to 100% ~Jentity lo the sequenca of E. dupr, E. hbwlyth and E roshkomkii laredo, deposited in GmBsnk with accession number IGcnBankZgUX]. [ G e n h n k m l and ICenBankAEuZZ&l mpcc(ively.

i\nswrrnt cfcmpcwm t%r mnwqet D M Ihe laKssment of competiuon of ohm non-law DNA prcmt in stool DNA M a with a larsct DNA in nested ~nultiplu PCR showed apened amplification without rny non-specific amplification.

ErPmatim cfmiunun nmber dEnornocbr cdr detsctabk by n d mubpkr POI I l~e nested multiplex PCR detened E. hurolyt~cn. E. d q w r .and E. mmhkolllnr DNA, wcn at the minimum parasite concentration tested (1000 parasites10 05 grams of fae- ,m) The detection limit of nated multipla PCR for E. haroryl~cii. E. duprr and E. mmhlmvsh~ was found to be rpprodmately 25 Enlrrnweba pmlowa cells, since 2.5 pl of lrmplate DNA (I000 paranite/lOO p1 ofTE buffer) gave positive signal (Figure I ) .

''w I ~ ~ d P C R l a ~ d m W w m n u n k r d L . m h k a u (EM), E (EH) and E drrpor (ED) c e k 3 0 S ~ o t ~ c o n r d s m ( s p c c l m s e t d c d r * l c h u n - fir dyutcd Enronaba cc(h currrpondlng to IDcclh (lane 11 I01 csUI (lane 2). lW cctb (lane 3). 10'cdh (hm 4). 18 [I (kina 5). and 10 C&S (lane 6) were sub/Med m DNA m-(da*adbyPCRampUikaUon AmpHRedpmbco *madymdbylpmu~declrophann Thesimd IhcaqMcxionpmduc~antndlatedonthckft(h~ Pllrs) hms 7, npuhn c o w d (PCR wkhout DNA)

- - - - - -- - -- --- - --

E s l n m i m o f n e a c d ~ P O P m d n c Q m u t d ~ v a h E histdycia E. dirpv ad E rpda 1000 cells of E. dirprr and E. m a M z spccin as the background allowed for the detection of a 0.01 cell of E. huwlyfiur; 1000 cells of E. hirwlpiu and E. mmhkmkr species as the background allwed for Ihl daeccion of a 0.001 cell of E. dspnr ; and 1000 alls of E. hirwrytu and E. dispr specin u the badqpund allowed for the dcIec- tion of a 0.1 cell ofE. m m W i (Figure 2).

cmrschcdr i~ themulac fna icd~POP The croso checking of the results of nested multiplex PCR was satisfactory as the same results were reproduced in randomly selected samples showing a m i d infection when subipaed to individual species specific PCR in sep araie tubes.

NcrtedmuhipkxPcn 7he nested multiplex PCR developed and evaluated in the present study showed that the size of diagnostic fragments of PCR products was dearly different for aU the threeEnra- moeba spcaes, the spedes.spedfic product size for E. histo- lytrcn, E. moshkmkii andE. dkprwas 439.553 and 174 bp respectively (Figure 3).

The nested multiplex PCR was performed on a total of 237 stool specimens including 202 stool spedmens positive for E. harolynur. E. dispar, or E. morhkouki~ by microscopy andlor culture, and 35 amoebae-negative control stwl specimens. The nested multiplex PCR was positive in 190 w t of 202 stool specimens that were positive for E. huw iyl~ca/E dupm/E. mmhlro~hi complex trophozoites/cysts by microscopy and/or mlture, thus showing a sensitivity of 94%. All the 35 negative conml stool samples were negative by nnted multiplex PCR thus showing a specificity oi 10096

The probability of negative nested multiplex PCR results in 35 control stool samples due to PCR inhibitors was ruled out by spiking the DNA of negative stool swimens wth sundard DNA of Enln& followed by nested multipla PCR amplification.

'The nested multiplo K R A m e d mono-infection with E. hulo&l~~l in 7.4% (15 of 202). E d q r in 41.5% (100 of 202) and E. mdhwshi in 1.0% (2 of 202) of stool wmpln. The PCR also detec~cd mued infections by both E. duplrr and E. mhbadn~ in 8.9% (18 of 202), E. lkpar and E. hirldytrco in 18.8% (38 of 202). and E. hbrolynca and E. mmhbanLit i n 1.0% (2 of 202) of stool samples. l hc test slmdcccctcd mired infections by all the threespe. d n ( E . ISL LO^. E. d q r and E , inmhbdnt) in 7.4% ( 1 5 of 202) of stool samples (Table I). lhe result of the nested muldplu PCR u compared with microvow and culture is also summariud i n Tabk I .

F i p m 3 D i f l e r N l ~ d s c L c t i o n d E ~ . E ~ a n d E d ~ W by nested mu+x PCR on stool umpkr. The E mashh- & (EM). E. h- (EH) and E drpcv (ED) bands are 553.439 and 174 bp, respectively. Lm-I. E mdka&i (mono k w n ) : LLw-i E rnorhkmakm. E ud L dupar ( w e d 1demian). Lane.). E h-o ud E dspor

jmlxed ~ h ~ o n ) : h n - 5 . E m & M and E ddapa (mixed ~ k t i o n ) . L u c 6 E hat- (mono ir(cction), bm-7 E d& por (maw, demon): land. 100 b DNA M d u ( b l a r e

PCR positivity 016. dirpar, E. hiswlytlut and E. moshkmsbir among the stool samples that were positive for E. hiswlyt- tw/E. dmrlE. mahltovsh~ bv miaoscow andlor culture is

TcchLob E. hirro)lah I1 EUSA Ihe TechLab E. huw1)ztcn I1 EUSA test was performed to detea E hrrml)zu coproantlgcn In 45 randomly selec~ed stool sam~les that were Dosltlve for E hlr&rwlE. d~suurl f rnorhkokkw complex by microscopy and/or ~ ~ h ~ r e .

Ihe Techlab E. hlrrolynr~~ I1 EUSA detened copmantigen 111 29 out of 4 5 (64.4%) stool specimens positive for E. hinolyttca/E. d&r/~ . m m h k o ~ l i n ~ m m ~ l a by microscopy andlor mlturc These 29 amoebic antincn msitive stool " , speclmenr included 4 specimens positive for E, hirrolyhca as mominfection 17 specimens positwe for E, hictolynw and F. r l ~ ~ p r r as m*pd.lnfection, 1 specimen pmitlve for t huwlyruu and E mahkorsh~ as mixed-infection, and 7 specimens positivr for all the three rpccia namely E, hi.+ wl)v~u, E mmhkm.r&it and E. d i c p as mixed-infenion hy thc nested muldplu PCR ('l'able 3).

CDmparison of results of nested multipla PCR and'l'ech- lab E hlrurEMlrm I1 EUSA test wrformed on 45 stool swcimens is summar id in the uble 3.

Dkuukn ' lo be able to drtect and distinguish E. hmailncn. E. dupar and E. m m h M i in stool samples is mrnnely important

TwdEnmrb. NO.OfYmp*tpohh.bynoad m mvbpb PCR

L " I M M ~ U 37 21 cw 171 99

70 4 0 . - L- - - - -

' T d ~ n b r d d ynph cdbcted tmn pa- m a h g 11WER lmphd. hdudmv h& mrh cmfhlnrs dpummrntlrul &r- = 1 720

newly described P C R techniques may yield uscful infor- mation especially in the field of molecular-based diagno. sis of intestinal amoebiasis.

Nested PCR waa used in the p m t study because it increases sensitivity 1181. ~ l i n i c i specimens such as slool often contain PCR inhibitors even after purification steps. The two mnds of P C R might have assisted in comp.nsat. ing the &em of inhibitors prcaent in clinical specimens. The first F C R produn maybe in UUJ Iowconcenlration for detection with cthidium bromide stained aels usinn a UV transilluminator. The detection limit of agarcme gel elec. trophoresis with ethtdium bromide atat; us~ngan IN lranollluminator~cawroximatelv 10 neof DNA 1191. The . . , " . .

lor accurate diagnosis of intestinal amoebiasis and for product of fint P C R may be just enough to provide ade. knowing the wuc pnvakncc of path-ic E. hirwlytlca in quate templates for h e &theis of second k~ product ihe communitv. Various DNA based molmlar methods in the nested reaction to be detected bvcthidium bromide have been evd;ated for accurate detection of E. humiytlm. staining. i duparor E, rnmhkmhi 12.6.9-161.

The nested multiplex K R was negative in 12 oul of 202 Ihe p m n l uudy, describes a new npsted multiplex PCR stool swcimens that were positive for E. histolvricalE. dir- strategy for specie+-specifir detection and differ&ttat~on par / ~ . ' m o r h ~ ~ comple; trophozoites/cysts'by micros- ,( 1. hrctdyaur. E d w r and d nwshhxbt~ DNA dtrectly copy and/or culture The negauve result due to ~nh~bltton in thc wool samples dpa~ents of P C R ln a11 these 12 stool samples was led out bvsak- . .

ing with standard DNA of ~n&nocba followed by k i ted Rcrmtly a single mund K R to detect I:. huldyticn. E. du- multiplex PCR amplification. rvr and E, mmhkmh~ in stool samples has been repned ?v t f a m h et a1 (171, the wudy reponed that out of 27 The negative P C R w l t in hese I2 stool samples may be uool sampks poaitivc for €nu& spp, by micrwopy due tobe presence of other Enramabn specie; However. ~nlv 7 were succcrrhrllv identified at srxcies level bv PCR we feel thatthisw~~)sition needsto be Droven bv funher . . ehrh Included I poailve for E, huw(m and 6 fo;~, du- development of molecular tools to conkm the presence >ti. hut no amplificalion of E, rnmhkov*l~ was observed In of othcr Enumoebn species commonly found in humans. I lha~ population ( 171 In contrast. our study has shown such as E. wh. E. harrmann~ or othcr similar looking Enm 'hc presence of E. nahLwsm in the Indian population. &I species. Xl l then these negative results may be .hc negative r m l t for E. mmhlto&t In lhai population Imputed to the sensitivity limitation of thc nated multi- .nav be attributed to thc small sdmple size used in the plex PCR technique. .ardy A detailed comparative study between these two

l ~ l . b m p . & a d r a k d l a a d m u h p * ~ ~ ~ ~ ( C ~ C h J n - ) u J T r h L b L ~ I I E U Y ~ l t Mamda*ool-

h n t d m*ph PCR ra* rcchlrb E -0 I1 uml ~pcmrn m nruh. T d m ol ~ m m - - - - - - - - - - -

m- - - NcpDn - - - -- -. -

- ~ n a . - l 0 # I L k + = a I m o m - ) 4 1 6 '-(maodmxm) 0 0 0 j a ~ + ~ n * . ~ m ~ ( - w o 1 I

6 1 P D I + E ~ ~ ~ r d ) I7 I I8 L-+L-(nhnd) I I i w L - - m N d l 1 I 10 '*ra D 0 0 - - - -- 'ad I* I* 45

- r ~ l ~ ~ ~ 1 . r r ~ 1 0 d m n ~ E ~ W ~ ~ m n m l ~ ~ b u Q . ~ ~ ~ ~ *ttnr.dE hbm@m

In he prescnl study by using nested multiplex PCR i t was shown that the raQ of mono infection with E. dispr was Ole highest E. diswr was demonmated in LOO out of 202 r t w l rrmplca (49.5%) amongu patients attending IIP- +lER hospital. The sardy also shorn that the rate of co- 111fmion with E. huwfyaca and E. dupar was the highest ( 18.8%) u compared to both, E. dispar and E. morhlwrlni 18.9%). and E. hirwlyncd and E. dkmhi (1.0%). The ~~cuncnce of co-infmion with E. dupm and E. hirwfyhm 111 the stool spccimena has been documented by men1 irudica reported earlier [3,10,12.20,21~.

Vested multiplex PCR was positive for E , hirwlytico DNA In 6 stool specimens which were negative for coproanti- gcn by'l'cchlab EUSA. lhe possible reason for such occult ~nfection (PCR-positive. EWSA-negative stool speamens a,r t. hlnwly~~d) may be due to degradation of lenin anti- pn in stool specimen due to prolonged storage (approximately 30-60 days) at -20'C pnor to testing thus ~nulting in a -tin test for amoeb~c coproantigen by !he EUSA

In h e p e n t study the overall conelauon between the r~.iultsof nested mulupla PCR and that oll ichlab E. hu- :,l,ma ll ElJSA to d d m I-:. hiswfyfia in stool specimen *as greater than '>O%. 'lhrs agreement between hex two Irchn~quesshows rlearly that either of the techniques may hc used alone to y~eld an accurate asscssmmt of the prer- F ~ ~ r c of I<, huio1yht.a In a stool specimen. but not for the J~trroon of erther E. &par or 8, mhkm&u In stool spa- ilnrns hy lrrhlab E. huwlyncn I1 ELIM.

he tnabrl~ty of Iechlrb hufnlytun II tIJSA to d d m ilhcr t duplr or E mmhCorhr In stool spcctmena u Ihc ~i)ted dsadvantage of the test Ihc nesled multtpla PCR II the other hand appears to be more useful for s~mulu- in,u dentrun of all the three spec~nt huwfyhm. E n-1 and t dupn whm performed d~redy on the 5rrwl mpcrtmcru Ihrr 1s the matn advantage of ths tesb rnd s of tmponancc due to the fact that t h m 1s an brreulng dorumcnutron of boll1 t m h h h t and E

J WI from d~licrcnt paw of rhc world ~1.6.9.11 171 ~tclud~ng from Puducherry thc southern unlon terntory

,kctron amongrt the prtrnts howed an Increased p o s ~ ~ l ~ ~ l r t y of faulty d~agnosu when thc ~dcnuficauon of E lirol)*ua was b l v d pnmar~ly on morpholw by mbcro-

~uprc cumlnatton of stool Ihc hlgh PCR posrunry of E ~ c " h k 4 r among rhc study populauon supporn the urn that humam arc a true host for th~s hrr-lmng 'nl~wba (21

ltlv inability to distinguish E. hulolyout from h x c of "~~rpholylicrlly similar E. dupar or E. moshlmusht in he

stool samples is the main limitation of microscopy or culture. As shown in the present study only 34.6% of 202 stool specimen8 positive for E. historn. E. dirpar or E. nwshkhkii com~loarophozoim/cysu by either micros- cow or culture were actualtv E. hivolrtiur and the remain- . , inn maiorin of stool smimena were positive for E. dkwr a n a o r ~ . iorhlmysk~i.~ln the aixfncc-of w auch as the nested multiplex PCR or EWSA for specific detection of E. hiswhicn. the maioritv of susoected infeaiona would haw bcen'wrongly dLgn&ed a; E. h is torn infection and treated unnecessarily with anti.amoeb'~ drugs. We therefore, recommend the use of the nested multiplex PCR for simultaneous detmion and accurate identification of all the three Enfnmoeba species in stool specimens.

Conclurion The present study reports a new nested multiplex PCR strategy for species specific detection and differentiation of E. hiswlyuar, E. dupr and E. mo&mkii DNA in s-l specimrns. l hc test is highly specific, sensitive and also rapid; results of the test are available within 12 hours of receipt of stool specimens.

Methods s o r n y c k h A total of 1.755 stool samples were collected in the pment study during a study period of two years from luly 2004 to luly 2006. This included 1,720 stool specimens collmed from patients attending 1lPMF.R hospital. Puducheny, India, with complaints of gastrointestinal discomfort. It also included 35 wl samples, as control sampln collected from healthy persons as well as pati;nts with ocher intestinal infections. 1he 35 control a m l samples included 15 stool samples negative for com- man enteric oarhopem from healthv m m . 6 samples . " , . pwove for cntenc bactena bv bacterial culture. 10 posl- uvc for Lurdvl lnrnnnalu cysts and 4 for eggs of hccmrc lvnbrimtda All these 35 stool samples were negative for E, hualpra/E. dlrplr/E. mmhkmhi~ complaq%ts and tro- phomites by microstopy and mltun.

Fresh unpresewed stool samples were collected in rlerile capped containers for examination by microscopy and culture for E. huwlyturr/E dirpar/E. morhhhr complex. Aliquotsof fnlh unpresewed stool samples were stored at -20'C until used for PCR and EIISA tau.

mcmcopk eromhmtk.n of nod BMh rrlirw and iodine wet mounts of msh unprescwed stool samples were cumined micrmmpically for demon- unting E. hnrdynca/E. dirpurlE. m o s k d i i complex cysts and tmphozoites as previously d d b c d 1221. Brielly. saline WR mounts were made by mixing approximately 2 mg of stool with a drop of physiological saline on a glass microstope slide and placing a corn slip over the stool

suspension. Similarly, iodine wet mounts were prepared by adding appmximatcly 2 mg of scml to a drop of I.ugol's iodine (diluted 1:s with distilled water) on a glass ~nicmrcope slide and placing a cover slip on the stool suspension. These wet mounts were microscopically exam- ined initially by using a low-power (10.) objective and then u d q a hi@-pown (401) objective of a compound l~ght micnncope. 'h wet mount was read in approximately 5 min to view at least 100 fields per slide. Each r l m l wmple was screened by at least three well trained micmrcop~sts hefore reporting negative results in our lab- ,,ratory.

S l d album \ lml samples were cultured for Enramabn species in I n c k e ~ (LA) medium (NIH mod~dca~~on of heck and i~rbohlav's medium) wlthln 6 h of collection aa previ. ously dacrlhed (23.241.

N u t a d mhlpkx- pdpmmaa eheln rrsctlon (PUI) - d m - w Ihc DNA was tsolaled from stool specimens by a cetyltri- methylammon~um bromide (CTAR) extradon method mcdified from prev~ously dncrikd method for DNA i s iruon from ~n wtro cul~resof Enramdm 1251. Briefly. 50 mg of nml spnmcn, was dispersed in 250 pl of lysis hillier (0 25% uxi~um dcxlecyl ~ulfate in 0.1 M EUTA, pl l

( I ) , and 100 WL/ml of proteinase K was added:Ihe lyratc ,%as amubaled a 55'C for 2 houn, lhcn 75 pl of 3.5 M \aCI follwed hy42 p1 of 10% ClABIO 7 M NaCl (heated :#I 55-C) w u added. After h e cornpownu were mired. ' ! ~ e urnplc was ~ r m b a t d 41 65'C for 30 mln. 'lhis was 1, mllowed by rxtractiona with equal volumes ofchloroform rnd thm phcnol-chlomfom-immyl alcohol. and the :IVA was prrnpilatcd wtth ace cold cthrml. The dried 'IYA y l k t war dissolved in stcnlc dintlled water and .?.~ssed over a L)NA clean.up spin column (Ranglore .mr i KI-62. Bangalore) 'Ihe DNA was finally eluted 'rum h e ~ 1 1 1 column In 100 )11 of'lns-EUIA [IE) buffcr. : 5 p1 of thu DNA solution war uxrd in thc PCR reaction

Quami$aGm of DNA in aod q e h c n Quantification of DNA in spin column purified DNA auact hom stool specimens was daerrnined by UV absorbance using a Cintra 5 double beam spectrophotom- eter. DNA yields were ulculatcd on the basis of U V absorbance M dilution.The purityofthe nucleic acid in the samples was estimated by the ratio of readings at 260 nm and 280 nm (OD ,JOD ,,). lhc quantification of DNA was done only for representative stool specime~ to know the DNA yielding capacity of the CTAB-DNA extraction method and also to estimate the pulity of extracted DNA for its suitability to be used in PCR

Rmcr drripn The genus specific primen were designed using nucleotide sequences of 16s- like rRNA gene of E. dicpar, E. hiswlyncn and E. m o s h ~ h ~ Laredo dcpooited in CenBank with accession number [ C e n B a n k : ~ 1 , IGen- B a n k : W 1 and (GenBan-1 respecuvely. Ihe comparison of all the three 16.5-like rRNA gene sequences of E. dirpar, E, hrtwlyucn and E , morhbowkii laredo rpvealed significant differences enough to design species specific primen. The primers were designed using Primer 3 on.lincsohare [261.'Ihe primer sequences used for nested multipla PCR are shown in table 4

R e r voLdomn 'The primer sequences designed for E. moshkomlnr. E. h b Iplca. and E, dirpm were subjmed to a Basic Local Align- ment Search 'lbol (BLAST) in the genome database of all organisms 1271 and were found to be swific for the nu*.

Ihe ampl~fied PCR produns of all the three species in r~ool sampla were confirmed by getting both the strands of DNA sequenced on ABI PRISM 377 sequencer (Indian Institute of Science. Bangalore, India). Briefly, the ABI PRISM 377 DNA sequencer auwmatically analyzes DNA lno~c~ les labeled wlth multiple fluorescent dyes. Aher samples are loaded onto the system's vertical gel, they

cm cheddng Phe m h dmrted nwhipkx Po1 some o f the r e p m n t a t i v e s t m l s a m p l e showing mixed ~nfect ion b y nested m u l t i p l u PCR were selected randomly and s u b w e d to species specific individual nested I'CR. w i th species specific p r i m e n for each species, in scp "rate PCR tubes.

Tachlob E. W EUU (cn

Ihe'~cchLab E. hutolyrlcn I1 EIISA test w u performed to Jctrrt E, huwlp~rd copmantigen i n 45 randomly selected <tool samples, poritive for I:, htsrolpca/E. diqar/E. morhk- 2:7Cl1 romplex. by m i c r w o p y and/or culture, and also pnsllive for F hrr1D))llcrl E d~spnr and E. m r ~ i DNA av n n t e d multiplex PCR. ' f i e Techhb E. h u w l y t u II il l% t n t was performed as per the i m t n r d o n s o f the ni.~nufarrurer ' lhe ki t was generously given for the put. ;v,se o f rcwarch by Tffhlah. Inc (Hlackshurg Va.).

Authon' contribubbns hk: camed out h e e x ~ r i m e n l a l works, and drafted the nlanusrnpt X P supervlwd and coordinated the study. ~ n d helped I n thc complrt ton the manuscript,

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~ ~ ~ - d = ~ $ 1001. U:r)3-297.

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la kwrr TO. SK. ~bmrrl JF. oaudhy H. FM, BS: ~ d a N c r t o d . ~ K R c o ~ a k O u t . - J ffi Mcrobd 1W7. >f:2M3-Mlb.

mCq Pnrr 2m(tS.I45.15. m. -- oh s - . ~ L nomcn.~- JL b.

~ l l . L m u h O n ~ ~ n Q ~ ~ ~ c ~ I n ~ w i t h ~ mmeb - mcm+mhrir %%ion childrrn. M M e d R n l997~97.uIuII5111.

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Common dimma, simple test

Reliable diagnosls of amoebic liver abscess (ALA) - a common parasitic infection - requires extracting blood or pus from the abscess, an invasive procedure. Now, microbiologist Sub- hash Parija at the Jawaharlal Institute of Postgraduate Medical Education and Research (Jipmer), Pondicherry, has shown tor the first time that Enta- moeba hystolytica - the parasite that CeUS8S the d i e - m y .kpg be detected thmugh a simple urine teat Pent;+ and hls L\.::@&&- Krlshna Ktubirw, have damonstrated that the genetlc material of this parastte pass- es through the human kidney and is excreted in urine. Doctors estimate that amoebiasis annually causes ill- ness in some 34 to 50 million people worldwide and leads to 40,000 to 100,000 deaths, mainly from ALA.The Jipmer study, published in the journal BMC Micmbiollogy, m y pave the way for a new way to diagnose ALA. Monitoring the excretion of the parasite's DNA in urine may also pcPvld.awaytotracktheFssQonse of a patlent who has been given metmnidarde, the standard treatment for the lnfectlon.

addendum - shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/951/18/18... · 2012. 7. 11. ·...

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